Purification of DS-DNA using heteropolymeric capture probes and a triplex, quadruplex or homologous duplex binding mechanism

ABSTRACT

A purification method includes bonding a probe to a target in a sample to form a complex, and separating the sample from the complex to separate in a sequence specific manner the target from the sample. The complex, which is immobilized on a support, is a duplex, triplex or quadruplex formed by Watson-Crick complementary base interaction or by homologous base interaction, provided that when the complex is a duplex and the heteropolymeric probe sequence is antiparallel to the heteropolymeric target sequence, the heteropolymeric probe sequence is bonded to the heteropolymeric target sequence by homologous base interaction, and provided that when the complex is a triplex, the complex is free of recombination proteins. A kit for performing the method includes the support and the probe.

BACKGROUND OF THE INVENTION

[0001] 1. Field of Invention

[0002] The invention relates to nucleobase binding in complexes, such asduplexes, triplexes and quadruplexes, and more particularly to methodswherein such complexes are formed by specific binding betweensingle-stranded or double-stranded nucleobase-containing capture probesand single-stranded or double-stranded nucleobase-containing targetsequences.

[0003] 2. Description of Related Art

[0004] The Watson-Crick model of nucleic acids has been the acceptedstandard in molecular biology for nearly fifty years. As recounted byJames Watson in his book entitled “A Personal Account of the Discoveryof the Structure of DNA,” (1968), the Watson-Crick model, which wonWatson and Crick the Nobel Prize, arose from the ashes of theirabandoned theory that bases bind to like bases on opposing strands(Watson at p.125). Watson described how he abandoned his “brieflyconsidered like-with-like pairing” model when he realized the advantagesof a model based on A:T and G:C binding. Id.

[0005] Although antiparallel nucleic acid duplexes first suggested byWatson and Crick are the most widely studied type of multiple-strandnucleic acid structures, it has been discovered that nucleic acids alsoform triplex structures and quadruplex structures under certainconditions.

[0006] Until recently, binding among three nucleic acid strands to forma triplex was widely believed to be confined to very limited species ofnucleic acids (e.g., polypurine or polypyrimidine sequences). See, e.g.,Floris et al., “Effect of cations on purine-purine-pyrimidine triplehelix formation in mixed-valence salt solutions,” 260 Eur. J. Biochem.801-809 (1999). Moreover, canonical triplex binding or hybridization wasthought to be based on Hoogsteen binding between limited varieties ofadjacent nucleobases, rather than Watson-Crick base pairing. See, e.g.,Floris et al. and U.S. Pat. No. 5,874,555 to Dervan et al. However, someof the inventors have recently disclosed in several patent applicationsthat specifically bound mixed base sequence triplex nucleic acids basedon Watson-Crick base pairing can be created and used as the basis for ahighly accurate and sensitive assay for specific binding. See U.S. Pat.Nos. 6,420,115 and 6,403,313.

[0007] Zhurkin et al., 239 J. Mol. Biol. 181 (1994) discloses thepossibility of parallel DNA triplexes; however, these triplexes are saidto be created by the third strand binding in the major groove of theduplex in the presence of recombination proteins, such as RecA protein.

[0008] As has been the case with triplex nucleic acids, the conventionalwisdom regarding quadruplex nucleic acids has been that such peculiarstructures only exist under relatively extreme conditions for a narrowclass of nucleic acids. In particular, Sen et al. (Nature 334:364-366(1988)) disclosed that guanine-rich oligonucleotides can spontaneouslyself-assemble into four-stranded helices in vitro. Sen et al.(Biochemistry 31:65-70 (1992)) disclosed that these four-strandedcomplexes can further associate into superstructures composed of 8, 12,or 16 oligomers.

[0009] Marsh et al. (Biochemistry 33:10718-10724 (1994), and NucleicAcids Research 23:696-700 (1995)) disclosed that some guanine-richoligonucleotides can also assemble in an offset, parallel alignment,forming long “G-wires”. These higher-order structures are stabilized byG-quartets that consist of four guanosine residues arranged in a planeand held together through Hoogsteen base pairings. According to Sen etal. (Biochemistry 31:65-70 (1992)), at least three contiguous guanineswithin the oligomer are critical for the formation of these higher orderstructures.

[0010] It has been suggested that four-stranded DNAs play a role in avariety of biological processes, such as inhibition of HIV-1 integrase(Mazumder et al., Biochemistry 35:13762-13771 (1996)), formation ofsynapsis during meiosis (Sen et al., Nature 334:364-366 (1988)), andtelomere maintenance (Williamson et al., Cell 59:871-880 (1989)); Baranet al., Nucleic Acids Research 25:297-303 (1997)). It has been furthersuggested that controlling the production of guanine-rich quadruplexesmight be the key to controlling such biological processes. For example,U.S. Pat. No. 6,017,709 to Hardin et al. suggests that telomeraseactivity might be controlled through drugs that inhibit the formation ofguanine quartets.

[0011] U.S. Pat. No. 5,888,739 to Pitner et al. discloses that G-quartetbased quadruplexes can be employed in an assay for detecting nucleicacids. Upon hybridization to a complementary oligonucleotide, theG-quartet structure unfolds or linearizes, thereby increasing thedistance between donor and acceptor moieties on different parts of theG-quartet structure, resulting in a decrease in their interaction and adetectable change in a signal (e.g., fluorescence) emitted from thestructure.

[0012] Silica materials, including glass/silica particles, glass/silicagel, mixtures of the above, and diatomaceous earth, have been employedin combination with aqueous solutions of chaotropic salts to separateDNA from other substances and render the DNA suitable for use inmolecular biological procedures. See U.S. Pat. No. 5,075,430, Marko etal., Anal. Biochem. 121, 382-387 (1982) and Vogelstein et al., Proc.Natl. Acad. Sci. (U.S.A.) 76, 615-619 (1979). With reference toseparation of RNA using silica materials and chaotropic agents, see U.S.Pat. No. 5,155,018 to Gillespie et al. These particle matrices arecapable of reversibly binding nucleic acid materials when placed incontact with a medium containing such materials in the presence ofchaotropic agents. Such matrices are designed to remain bound to thenucleic acid material while the matrix is exposed to an external force,such as centrifugation or vacuum filtration, to separate the matrix andnucleic acid material complex from the remaining media components. Thenucleic acid material is then eluted from the matrix by exposing thematrix to an elution solution, such as water or an elution buffer.Typically, these methods are carried out to obtain either a highlypurified quantity of a single target nucleic acid segment such as wouldbe found in a PCR reaction or plasmid purification, or obtaining wholecell DNA or RNA sufficiently free of contaminants for molecularbiological applications. The main drawback is that in a mixture ofnucleic acids, there is no sequence specificity to select for or againstthe increased quantity of a single nucleic acid segment. This desiredtarget nucleic acid along with all other sequences of nucleic acid willbe captured and bind tightly to the particles without any preference.

[0013] U.S. Pat. No. 5,912,332 to Agrawal et al. discloses a method forthe purification of synthetic oligonucleotides, wherein the syntheticoligonucleotides hybridize specifically with a desired, full-lengtholigonucleotide and concomitantly form a multimer aggregate, such asquadruplex DNA. The multimer aggregate containing the oligonucleotide tobe purified is then isolated using size-exclusion techniques. However,this method requires a sequence defined multimerization domain to allowfor simultaneous aggregate formation and hybridization to the targetoligonucleotide.

[0014] Ito et al. (PNAS 89 (1992) 495) and U.S. Pat. No. 6,319,672 toCrouzet et al., describe the use of biotinylated oligonucleotidescapable of recognizing the homopurine/homopyrimidine sequence in aplasmid and of forming a Hoogsteen-type triple helix. The complexesformed are then brought into contact with streptavidin-coated magneticbeads or column chromatography matrix. Interaction between the biotinand the streptavidin then enables the plasmid to be isolated by magneticseparation of the beads followed by elution or separation off the columnby standard chromatography. However, this method has some drawbacks. Inparticular, a capture sequence is required to be inserted into theplasmid, adding to the complexity of the method.

[0015] Despite the foregoing developments, a need has continued todevelop rapid and convenient means of capturing single stranded ordouble stranded mixed base sequence nucleic acids.

[0016] All references cited herein are incorporated herein by referencein their entireties.

BRIEF SUMMARY OF THE INVENTION

[0017] The invention provides a complex comprising: (1) a probecontaining a heteropolymeric probe sequence of nucleic acids or nucleicacid analogues; and (2) a target containing a heteropolymeric targetsequence of nucleic acids or nucleic acid analogues, wherein: (a) atleast one of the probe and the target is purified or synthetic; and (b)the heteropolymeric probe sequence is bonded to the heteropolymerictarget sequence by Watson-Crick complementary base interaction or byhomologous base interaction, provided that when the complex is a duplexand the heteropolymeric probe sequence is antiparallel to theheteropolymeric target sequence, the heteropolymeric probe sequence isbonded to the heteropolymeric target sequence by homologous baseinteraction, and provided that when the complex is a triplex, thecomplex is free of recombination proteins.

[0018] Further provided is a method for capturing and separating atarget from a sample, said method comprising: (1) providing the samplecomprising the target, wherein the target contains a heteropolymerictarget sequence of nucleic acids or nucleic acid analogues; (2)providing a probe comprising a heteropolymeric probe sequence of nucleicacids or nucleic acid analogues; (3) bonding the heteropolymeric targetsequence to the heteropolymeric probe sequence to provide a complex; (4)attaching the probe to a support before, during or after the complex isprovided; and (5) separating the sample from the complex to separate thetarget from the sample, wherein the heteropolymeric probe sequence isbonded to the heteropolymeric target sequence by Watson-Crickcomplementary base interaction or by homologous base interaction,provided that when the complex is a duplex and the heteropolymeric probesequence is antiparallel to the heteropolymeric target sequence, theheteropolymeric probe sequence is bonded to the heteropolymeric targetsequence by homologous base interaction, and provided that when thecomplex is a triplex, the complex is free of recombination proteins.

[0019] Still further provided is a kit for separating a target from asample. The kit comprises a support and a probe, wherein the probe isadapted to form the novel complexes of the invention, and is attached tothe support or adapted to attach to the support during performance ofthe method.

[0020] Also provided is a method for assaying a target, the methodcomprising: (1) providing a sample comprising the target containing aheteropolymeric target sequence of nucleic acids or nucleic acidanalogues; (2) providing a probe containing a heteropolymeric probesequence of nucleic acids or nucleic acid analogues; (3) providing ahybridization mixture comprising the target, the probe, water, and abuffer; (4) incubating the hybridization mixture for an incubation timeeffective to bind the heteropolymeric target sequence to theheteropolymeric probe sequence to provide a complex; and (5) detecting asignal correlated with binding affinity between the probe and the targetto assay the target, wherein the heteropolymeric probe sequence isbonded to the heteropolymeric target sequence by Watson-Crickcomplementary base interaction or by homologous base interaction,provided that when the complex is a duplex and the heteropolymeric probesequence is antiparallel to the heteropolymeric target sequence, theheteropolymeric probe sequence is bonded to the heteropolymeric targetsequence by homologous base interaction, and provided that when thecomplex is a triplex, the complex is free of recombination proteins.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

[0021] The invention will be described in conjunction with the followingdrawings in which like reference numerals designate like elements andwherein:

[0022]FIGS. 1A, 1B, 1C, 2A, 2B, 3A, 3B, 4, 5A, 5B, 6, 7, 8, 9, 10A, 10B,11A, 11B, 12A, 12B, 13A and 13B are composite graphs of fluorescentintensity plotted as a function of wavelength for each sample analyzed;

[0023]FIGS. 14A, 14B and 14C are composite graphs of fluorescentintensity plotted as a function of time for each sample analyzed;

[0024]FIG. 15 is a schematic view of dsDNA coupled to a solid support asa triple helix with a single-stranded oligonucleotide probe;

[0025]FIGS. 16A, 16B and 16C are serial views of a triplex affinityplate of the invention being used to purify target DNA;

[0026]FIGS. 17A, 17B and 17C are serial views of an another triplexaffinity plate of the invention being used to purify target DNA; and

[0027]FIG. 18 is a schematic view of a hairpin oligonucleotide probecovalently coupled to a solid support forming a triple helix with assDNA.

DETAILED DESCRIPTION OF THE INVENTION

[0028] The invention flows from our elucidation of the specific bindingproperties of heteropolymeric nucleic acid strands. We have previouslydisclosed the specific binding of a heteropolymeric strand to duplexnucleic acid and the specific binding of duplex nucleic acid to otherduplex nucleic acid. We now disclose that heteropolymeric nucleic acids(and/or their analogues) can specifically bind to each other byhomologous base bonding as well as by Watson-Crick base interaction, andthat base bonding is not limited to strands having antiparalleldirectionality relative to each other. Thus, heteropolymeric nucleicacids (and/or their analogues) can specifically bind to each other withparallel or antiparallel directionality, wherein the bases bond byhomologous base bonding and/or Watson-Crick base bonding rules.

[0029] The invention is more than merely the disclosure of the use ofunorthodox binding properties of nucleic acids. The inventionencompasses the formation and use of novel compounds in a method forcapturing nucleic acids, as well as methods for the analysis of nucleicacids, diagnostic methods, therapeutic methods, prophylactic methods,gene therapy and genetic engineering.

[0030] The invention encompasses the formation and use of novel duplex,triplex and quadruplex complexes of nucleic acids (and/or analoguesthereof).

[0031] Nucleic acid strands have inherent directionality. Theconventional wisdom holds that strands of opposite directionality, i.e.,which are antiparallel in their orientation to one another, can form aduplex through Watson-Crick complementary binding of their respectivebases.

[0032] Certain duplexes according to the invention, on the other hand,comprise two strands of nucleic acid (and/or nucleic acid analogues)hybridized in parallel relation to one another, wherein specific bindingis either through homologous base pairing or Watson-Crick base pairing.Conventional wisdom holds that such duplexes do not exist, or at leastwould be extremely unstable due to, e.g., backbone irregularitiesnecessitated by the conformational requirements of parallel basebonding. Even more surprising is our discovery that under appropriatehybridization conditions, homologous bonding demonstrates specificityand stability rivaling that of Watson-Crick complementary antiparallelduplex.

[0033] The invention also encompasses duplexes containing two strands ofnucleic acid (and/or nucleic acid analogues) hybridized in antiparallelrelation to one another, wherein specific binding is through homologousbase pairing.

[0034] As used herein, the terms “Watson-Crick base pairing”,“complementary base pairing” and the like are intended to definespecific association between opposing or adjacent pairs of nucleic acidand/or nucleic acid analogue strands via matched bases (e.g., A:T; G:Cand/or A:U). In the context of non-canonical complexes described herein,including parallel duplexes, parallel and antiparallel triplexes, andparallel and antiparallel quadruplexes, terms like “Watson-Crick basebonding” and “complementary base bonding” are intended to denote bondingbetween A and T, A and U and/or G and C, but not necessarily in theedgewise, planar conformation first described by Watson and Crick. Inaddition to the conventional binding motif first proposed by Watson andCrick (the “W-C motif”), and conformational variants thereof encompassedby the foregoing definition of Watson-Crick base bonding, the presentinvention encompasses complexes formed by homologous base bonding. Inhomologous base bonding, bases bond specifically with identical basesrather than complementary bases. Thus, in the “homologous motif”,homologous base pairs include A:A, G:G, C:C, T:T and U:U.

[0035] The binding by the bases of nucleic acid strands is affected orconditioned by a number of factors, particularly the binding potentialof the strands pursuant to either the W-C motif or homologous motif, andionic conditions (e.g., salt concentration and/or type). Saltyconditions tend to favor the formation of Watson-Crick bonding overhomologous bonding. Homologous motif quadruplexes are favored over W-Cmotif quadruplexes under identical buffer conditions probably becausethe localized environment can become relatively low-salt, based on thepresence of the charged backbones of the two duplex nucleic acids.

[0036] Each strand in a complex of the invention can comprise anysequence of nucleobases and/or nucleobase analogues, provided thenucleobases are related to the nucleobases to which they are tospecifically bind by either the W-C motif or the homologous motif.Contrary to certain teachings of the prior art, the target and probeneed not be homopolymeric to achieve binding, even in the case oftriplex or quadruplex formation. Thus, in certain embodiments, the probenucleobases are arranged in a heteropolymeric probe sequence ofinterspersed purines and pyrimidines, and the target nucleobases arearranged in a target sequence at least partially complementary orpartially homologous to the probe sequence. For example, the probesequence can contain 25% to 75% purine bases and 75% to 25% pyrimidinebases in any order. Complexes of the invention can form fromheteropolymeric sequences, which as defined herein, means sequencescontaining at least one purine nucleobase or purine analogue and atleast one pyrimidine nucleobase or pyrimidine analogue in at least theirhybridizing segments. Heteropolymeric sequences preferably lackhomopolymeric fragments greater than 5 bases long. Other nucleobases arealso suitable for use in the invention, such as, e.g., syntheticanalogues of naturally occurring bases which have specific Watson-Crickand/or homologous binding affinities to other bases.

[0037] In addition to duplexes, complexes of the invention also includetriplexes and quadruplexes, wherein opposing heteropolymeric strands arelinked by Watson-Crick complementary bases or by homologous bases, andthe relative directionality of the bound sequences is parallel orantiparallel to one another.

[0038] A probe strand can specifically bind in the major or minor grooveof a double-stranded target. Further, the bases of a single-strandedprobe can interact specifically with bases on one or both strands of adouble-stranded target. Similarly, the bases of each strand of adouble-stranded probe can interact specifically with bases on one orboth strands of a double-stranded target in quadruplex complexes of theinvention.

[0039] In certain triplex and quadruplex embodiments, each nucleobasebinds to one or two other nucleobases. Thus, in addition to thetraditional duplex Watson-Crick base pairs and the duplex homologousbase pairs described above, such embodiments include the followingWatson-Crick base binding triplets: A:T:A, T:A:T, U:A:T, T:A:U, A:U:A,U:A:U, G:C:G and/or C:G:C (including C⁺:G:C, and/or any other ionizedspecies of base), and/or the following homologous base triplets: A:A:T,T:T:A, U:U:A, T:U:A, A:A:U, U:T:A, G:G:C and/or C:C:G (including C:C⁺:G,and/or any other ionized species of base).

[0040] Thus, in certain quadruplex embodiments wherein the probe isdefined as a duplex of a first and a second strand and the target isdefined as a duplex of a third and a fourth strand, it is believed thatthe bases of the first and third strands also bind to each other, inaddition to: (a) the binding between opposing bases of the first andsecond strands; (b) the binding between opposing bases of the third andfourth strands; and (c) the binding between opposing bases of the secondand fourth strands.

[0041] In certain embodiments of the triplex and quadruplex structuresof the invention, no binding sequence of bases is contiguous withanother binding sequence of bases. That is, there are at least threeseparate strands. Although folded conformations and the like (e.g.,hairpin turns, etc.) are within the scope of the invention, foldedportions of a single strand do not make the strand count more than oncetoward the minimum of three separate strands.

[0042] Complexes of the invention preferably do not rely on Hoogsteenbonding or G-G quartets for maintenance of the complex structure,although Hoogsteen bonding and/or G-G quartets may be present. That is,complexes of the invention do not require the narrow class of nucleicacid sequences to form the triplex or quadruplex structures, arepreferably substantially free of Hoogsteen bonding, and aresubstantially free of G-G quartets.

[0043] Each strand of the complex independently comprises a nucleic acidhaving a deoxyribose phosphate or ribose phosphate backbone (e.g., DNA,RNA, mRNA, hnRNA, rRNA, tRNA or cDNA) or a nucleic acid analogue.Preferred nucleic acid analogues contain an uncharged or partiallycharged backbone (i.e., a backbone having a charge that is not asnegative as a native DNA backbone), and include, e.g., PNA and LNA.Certain embodiments are free of PNA.

[0044] At least a portion of the complex is isolated, purified,artificial or synthetic.

[0045] In embodiments, a portion of the complex is a PCR amplifiedproduct.

[0046] The complexes of the invention can be present in solution, on asolid support, in vitro, in vivo or in silico. The solid support can beelectrically conductive (e.g., an electrode) or non-conductive. Inaddition, the complexes can be optically mapped or sequenced after beingelongated, as taught in U.S. Pat. Nos. 6,147,198 and 5,720,928 toSchwartz.

[0047] Complexes of the invention can be provided by a methodcomprising: (a) providing a hybridization mixture comprising a targetcontaining a heteropolymeric target sequence of nucleic acids or nucleicacid analogues, a probe containing a heteropolymeric probe sequence ofnucleic acids or nucleic acid analogues, water, and a buffer; and (b)incubating said hybridization mixture for an incubation time effectiveto hybridize said heteropolymeric target sequence to saidheteropolymeric probe sequence to provide the complex.

[0048] The hybridization mixture can include any conventional mediumknown to be suitable for preserving nucleotides. See, e.g., Sambrook etal., “Molecular Cloning: A Lab Manual,” Vol. 2 (1989). For example, themedium can comprise nucleotides, water, buffers and standard saltconcentrations. When divalent cations are used exclusively to promotetriplex or quadruplex formation, chelators such as EDTA or EGTA shouldnot be included in the reaction mixtures.

[0049] Specific binding between complementary bases occurs under a widevariety of conditions having variations in temperature, saltconcentration, electrostatic strength, and buffer composition. Examplesof these conditions and methods for applying them are known in the art.Our copending U.S. patent application Ser. No. 09/885,731, filed Jun.20, 2001, discloses conditions particularly suited for use in thisinvention.

[0050] Unlike many Hoogsteen-type complexes, which are unstable ornon-existent at pH levels above about 7.6, the complexes of theinvention are stable over a wide range of pH levels, preferably fromabout pH 5 to about pH 9.

[0051] Complexes of the invention can be provided for capture, analytic,diagnostic, therapeutic and/or engineering purposes. The complexes canbe used to analyze, diagnose and/or treat conditions associated withinfection by an organism or virus. The organism or virus can bequantitated, if desired.

[0052] Complexes of the invention can be formed under conventionalhybridization conditions, under triplex hybridization conditions, underquadruplex hybridization conditions or under conditions of in situhybridization. It is preferred that complexes be formed at a temperatureof about 2° C. to about 55° C. for about two hours or less. In certainembodiments, the incubation time is preferably less than five minutes,even at room temperature. Longer reaction times are not required, butincubation for up to 24 hours in most cases does not adversely affectthe complexes. The fast binding times of the complexes of the inventioncontrast with the much longer binding times necessary for Hoogsteenbound complexes.

[0053] The promoter in the hybridization medium is preferably anintercalating agent or a cation, as disclosed in U.S. patent applicationSer. No. 09/613,263, filed Jul. 10, 2000. The intercalators areoptionally fluorescent. The intercalating agent can be, e.g., afluorophore, such as a member selected from the group consisting ofYOYO-1, TOTO-1, YOYO-3, TOTO-3, POPO-1, BOBO-1, POPO-3, BOBO-3, LOLO-1,JOJO-1, cyanine dimers, YO-PRO-1, TO-PRO-1, YO-PRO-3, TO-PRO-3,TO-PRO-5, PO-PRO 1, BO-PRO-1, PO-PRO-3, BO-PRO-3, LO-PRO-1, JO-PRO-1,cyanine monomers, ethidium bromide, ethidium homodimer-1, ethidiumhomodimer-2, ethidium derivatives, acridine, acridine orange, acridinederivatives, ethidium-acridine heterodimer, ethidium monoazide,propidium iodide, SYTO dyes, SYBR Green 1, SYBR dyes, Pico Green, SYTOXdyes and 7-aminoactinomycin D.

[0054] Suitable cations include, e.g., monovalent cations, such as Na⁺(preferably at a concentration of 40 mM to 200 mM), K⁺(preferably at aconcentration of 40 mM to 200 mM), and other alkali metal ions; divalentcations, such as alkaline earth metal ions (e.g., Mg⁺² and Ca⁺²) anddivalent transition metal ions (e.g., Mn⁺², Ni⁺², Cd⁺², Co⁺² and Zn⁺²);and cations having a positive charge of at least three, such as Co(NH₃)₆⁺³, trivalent spermidine and tetravalent spermine. Mn⁺² is preferablyprovided at a concentration of 10 mM to 45 mM. Mg⁺² is preferablyprovided at a concentration of 10 mM to 45 mM. Ni⁺² is preferablyprovided at a concentration of about 20 mM. In embodiments, Mg⁺² andMn⁺² are provided in combination at a concentration of 1 mM each, 2 mMeach, 3 mM each . . . 40 mM each (i.e., 1-40 mM each).

[0055] The amount of cation added to the medium in which the complexforms depends on a number of factors, including the nature of thecation, the concentration of probe, the concentration of target, thepresence of additional cations and the base content of the probe andtarget. The preferred cation concentrations and mixtures can routinelybe discovered experimentally. For triplexes, it is preferred to addcation(s) to the medium in the following amounts: (a) 10 mM-30 mM Mn⁺²;(b) 10 mM-20 mM Mg⁺²; (c) 20 mM Ni⁺²; or (d) 1 mM-30 mM of each of Mn⁺²and Mg⁺². For quadruplexes, it is preferred to add cation(s) to themedium in the following amounts: (a) 10 mM-45 mM Mn⁺²; (b) 10 mM-45 mMMg⁺²; or (c) 10 mM-40 mM of each of Mn⁺² and Mg⁺².

[0056] Although not required, other promoters include, e.g., singlestranded binding proteins such as Rec A protein, T4 gene 32 protein, E.coli single stranded binding protein, major or minor nucleic acid groovebinding proteins, viologen and additional intercalating substances suchas actinomycin D, psoralen, and angelicin. Such facilitating reagentsmay prove useful in extreme operating conditions, for example, underabnormal pH levels or extremely high temperatures. Certain methods forproviding complexes of the invention are conducted in the absence ofprotein promoters, such as Rec A and/or other recombination proteins.

[0057] The invention provides a rapid, sensitive, environmentallyfriendly, and safe method for nucleic acid capture or assaying binding.The inventive assay can be used to, e.g., identify accessible regions infolded nucleotide sequences, to determine the number of mismatched basepairs in a hybridization complex, and to map genomes.

[0058] The inventive assay not only detects the presence of specificprobe-target binding, but also provides qualitative and quantitativeinformation regarding the nature of interaction between a probe andtarget. Thus, the invention enables the practitioner to distinguishamong a perfect match, a one base pair mismatch, a two base pairmismatch, a three base pair mismatch, a one base pair deletion, a twobase pair deletion and a three base pair deletion arising between asequence in the double-stranded probe or single-stranded probe and in asequence in the double-stranded or single-stranded target.

[0059] Embodiments of the invention comprise calibrating the measuredsignal (e.g., optical, fluorescence, chemiluminescence,electrochemiluminescence, electrical or electromechanical properties)for a first probe-target mixture against the same type of signalexhibited by other probes combined with the same target, wherein each ofthe other probes differs from the first probe by at least one base.

[0060] A calibration curve can be generated, wherein the magnitude ofthe measured signal (e.g., fluorescent intensity) is a function of thebinding affinity between the target and probe. As the binding affinitybetween the target and a plurality of different probes varies with thenumber of mismatched bases, the nature of the mismatch(es) (e.g., A:Gvs. A:C vs. T:G vs. T:C, etc. in the W-C motif), the location of themismatch(es) within the complex, etc., the assay of the invention can beused to sequence the target.

[0061] In embodiments, the signal measured can be the fluorescentintensity of a fluorophore included in the test sample. In suchembodiments, the binding affinity between the probe and target can bedirectly or inversely correlated with the intensity, depending onwhether the fluorophore signals hybridization through signal quenchingor signal amplification. Under selected conditions, the fluorescentintensity generated by intercalating agents can be directly correlatedwith probe-target binding affinity, whereas the intensity of preferredembodiments employing a non-intercalating fluorophore covalently boundto the probe can be inversely correlated with probe-target bindingaffinity. The fluorescent intensity decreases for non-intercalatingfluorophores as the extent of matching (e.g., the amount of matches vs.mismatches and/or the types of mismatches) between the probe and targetincreases, preferably over a range inclusive of 0-2 mismatches and/ordeletions, more preferably over a range inclusive of 0-3 mismatchesand/or deletions.

[0062] The invention enables quantifying the binding affinity betweenprobe and target. Such information can be valuable for a variety ofuses, including designing antisense drugs with optimized bindingcharacteristics.

[0063] The assay of the invention is preferably homogeneous. The assaycan be conducted without separating free probe and free target from thehybridization complex prior to detecting the magnitude of the measuredsignal. The assay does not require a gel separation step, therebyallowing a great increase in testing throughput. Quantitative analysesare simple and accurate. Consequently the binding assay saves a lot oftime and expense, and can be easily automated. Furthermore, it enablesbinding variables such as buffer, pH, ionic concentration, temperature,incubation time, relative concentrations of probe and target sequences,intercalator concentration, length of target sequences, length of probesequences, and possible cofactor (i.e., promoter) requirements to berapidly determined.

[0064] The assay can be conducted in, e.g., a solution within a well ormicrochannel, on an impermeable surface or on a biochip. In certainembodiments, the target is provided in the hybridization medium beforethe probe, and the probe is provided in dehydrated form prior torehydration by contact with the hybridization medium.

[0065] In certain embodiments, the inventive assay is conducted withoutproviding a signal quenching agent on the target or on the probe.

[0066] The invention obviates the need to denature the target prior toassaying. It is surprising that the inventors have been able tospecifically assay heteropolymeric triplexes and quadruplexes, whereinthe interaction between the probes and targets is based on Watson-Crickor homologous base interaction (at least in the sense that A binds to T(or U, in the case of RNA) and G binds to C), rather than the verylimited Hoogsteen model of complex hybridization of, e.g., Pitner etal., supra.

[0067] Suitable targets are preferably 8 to 3.3×10⁹ base pairs long, andcan be single or double-stranded.

[0068] Probes of the invention are preferably 2 to 75 bases long (morepreferably 5 to 30 bases long), and can be single or double-stranded.Thus, suitable probes for use in the inventive assay include, e.g.,ssDNA, RNA, ssPNA, LNA, dsDNA, dsRNA, DNA:RNA hybrids, dsPNA, PNA:DNAhybrids and other single and double-stranded nucleic acids and nucleicacid analogues having uncharged, partially-charged, sugar phosphateand/or peptide backbones. The length of the probe can be selected tomatch the length of the target.

[0069] The instant invention does not require the use of radioactiveprobes, which are hazardous, tedious and time-consuming to use, and needto be constantly regenerated. Probes of the invention are preferablysafe to use and stable for years. Accordingly, probes can be made orordered in large quantities and stored.

[0070] The complex is preferably detected by a change in at least onelabel. The at least one label can be attached to the probe and/or thetarget, and/or can be free in the test medium. The at least one labelcan comprise at least two moieties.

[0071] The label is preferably at least one member selected from thegroup consisting of a spin label, a fluorophore, a chromophore, achemiluminescent agent, an electro-chemiluminescent agent, aradioisotope, an enzyme, a hapten, an antibody and a labeled antibody.Preferably, the complex is detected by at least one emission from thelabel or by monitoring an electronic characteristic of the complex.

[0072] The labeled antibody can be, e.g., a labeled anti-nucleicacid/nucleic acid antibody, which can be labeled with a detectablemoiety selected from the group consisting of a fluorophore, achromophore, a spin label, a radioisotope, an enzyme, a hapten, achemiluminescent agent and an electro-chemiluminescent agent.

[0073] The complex can be detected under at least one varied condition,such as disclosed in U.S. patent application Ser. No. 09/490,273, filedJan. 24, 2000. Suitable varied conditions include, e.g., (a) a change innonaqueous components of the test medium, (b) a change in a pH of thetest medium, (c) a change in a salt concentration of the test medium,(d) a change of an organic solvent content of the test medium, (e) achange in a formamide content of the test medium, (f) a change in atemperature of the test medium, and (g) a change in chaotropic saltconcentration in the test medium. In addition, the varied condition canbe the application of a stimulus, such as, e.g., electric current (DCand/or AC), photon radiation (e.g., laser light), or electromagneticforce. The stimulus can be applied constantly or pulsed. Detection canbe accomplished through the use of a single varied condition, or througha combination of conditions varied serially.

[0074] The response of a characteristic of the complex in the testmedium to the varied condition or stimulus can be monitored to detectthe complex. The characteristic can be, e.g., electrical conductance orQ (a resonant structure of a transmission line or changes in phase oramplitude of a signal propagated in the transmission line in the testmedium).

[0075] In embodiments, the detection method comprises: (a) detecting asignal from a label, wherein the signal is correlated to a bindingaffinity between said probe and said target; (b) varying a condition ofa test medium; (c) detecting a subsequent signal; and (d) comparing thesignal and the subsequent signal. The varying and the detecting can berepeated at least once or performed only once.

[0076] The label is preferably a fluorophore. Both intercalating andnon-intercalating fluorophores are suitable for use in the invention.The fluorophore can be free in solution, covalently bound to the probeand/or covalently bound to the target. When the fluorophore iscovalently bound to the probe, it is preferably bound to the probe ateither end. Preferred fluorescent markers include biotin, rhodamine,acridine and fluorescein, and other markers that fluoresce whenirradiated with exciting energy. Suitable non-intercalating fluorophoresinclude, e.g., alexa dyes, BODIPY dyes, biotin conjugates, thiolreactive probes, fluorescein and its derivatives (including the “cagedprobes”), Oregon Green, Rhodamine Green and QSY dyes (which quench thefluorescence of visible light excited fluorophores).

[0077] The excitation wavelength is selected (by routine experimentationand/or conventional knowledge) to correspond to this excitation maximumfor the fluorophore being used, and is preferably 200 to 1000 nm.Fluorophores are preferably selected to have an emission wavelength of200 to 1000 nm. In preferred embodiments, an argon ion laser is used toirradiate the fluorophore with light having a wavelength in a range of400 to 540 nm, and fluorescent emission is detected in a range of 500 to750 nm.

[0078] The assay of the invention can be performed over a wide varietyof temperatures, such as, e.g., from about 2 to about 60° C. Certainprior art assays require elevated temperatures, adding cost and delay tothe assay. On the other hand, the invention can be conducted at roomtemperature or below (e.g., at a temperature below 25° C.).

[0079] The reliability of the invention is independent of guanine andcytosine content in either the probe or the target. In the traditionalW-C motif, since G:C base pairs form three hydrogen bonds, while A:Tbase pairs form only two hydrogen bonds, target and probe sequences witha higher G or C content are more stable, possessing higher meltingtemperatures. Consequently, base pair mismatches that increase the GCcontent of the hybridized probe and target region above that present inperfectly matched hybrids may offset the binding weakness associatedwith a mismatched probe.

[0080] The inventive assay is extremely sensitive, thereby obviating theneed to conduct PCR amplification of the target. For example, it ispossible to assay a test sample having a volume of about 20 microliters,which contains about 10 femtomoles of target and about 10 femtomoles ofprobe. Embodiments of the invention are sensitive enough to assaytargets at a concentration of 5×10⁻⁹ M, preferably at a concentration ofnot more than 5×10⁻¹⁰ M. Embodiments of the invention are sensitiveenough to employ probes at a concentration of 5×10⁻⁹ M, preferably at aconcentration of not more than 5×10⁻¹⁰ M. It should go without sayingthat the foregoing values are not intended to suggest that the methodcannot detect higher concentrations.

[0081] The ratio of probe to target is preferably about 1:1 to about1000:1.

[0082] Unlike certain prior art assays, the invention not only detectsthe presence of hybridization (i.e., binding), but also providesqualitative and quantitative information regarding the nature of bindingbetween a probe and target. Thus, the invention enables the practitionerto: (a) detect the presence of the target in the test medium; (b) detectallelic or heterozygous variance in the target; (c) quantitate thetarget; (d) detect an extent of complementarity (in the case of bindingin the W-C motif) or homologousness (in the case of binding in thehomologous motif) between the probe and the target; and (e) detecthaplotypes.

[0083] We have noticed that duplexes which complex parallel strands ofnucleic acid containing complementary base sequences bind to formtriplexes at a different rate and bind as a culmination of a verydifferent process than do bases in a double helix formed by nucleic acidstrands of opposite directionality. Strands of opposite directionality(i.e., antiparallel strands) readily present regularly spaced bases in aplanar orientation to the bases opposite with minimal backbonedistortion.

[0084] The various complexes of the invention comprise a probecontaining a heteropolymeric probe sequence of nucleobases and/ornucleobase analogues, and a target containing a heteropolymeric targetsequence of nucleobases and/or nucleobase analogues. The complex issynthetic or purified in that at least one of either the probe or thetarget is synthetic or purified. The backbone of the probe is adeoxyribose phosphate backbone such as in DNA, or a peptide-likebackbone such as in PNA, or is of some other uncharged or partiallycharged (negatively or positively) moieties.

[0085] In certain embodiments, the probe and target are single-strandedand the complex is a duplex. When said probe and target are a duplexthey have parallel directionality with W-C complementary or homologousbinding, or have antiparallel directionality with homologous binding.

[0086] In other embodiments, either the probe or the target issingle-stranded and the other of said probe or target is double-strandedand the resulting complex is a triplex. This complex can be free of PNA.

[0087] In certain embodiments, the triplex contains a heteropolymericprobe sequence parallel to a heteropolymeric target sequence, whereinthe heteropolymeric probe sequence is bonded to the heteropolymerictarget sequence by homologous base binding or Watson-Crick complementarybase binding. In certain other embodiments, the heteropolymeric probesequence is antiparallel to the heteropolymeric target sequence and theheteropolymeric probe sequence is bonded to the heteropolymeric targetsequence by homologous base binding or Watson-Crick complementary basebinding.

[0088] In certain embodiments of the triplex complex, the targetincludes a first strand containing a heteropolymeric target sequence anda second strand containing a second heteropolymeric target sequence thatis Watson-Crick complementary and antiparallel to the firstheteropolymeric target sequence. The heteropolymeric probe sequence isbonded to the first heteropolymeric target sequence by homologous basebonding and is also bonded to the second heteropolymeric target sequenceby Watson-Crick complementary base bonding.

[0089] In certain other embodiments of the triplex complex, the targetincludes a first strand containing a heteropolymeric target sequence anda second strand containing a second heteropolymeric target sequence thatis Watson-Crick complementary and antiparallel to the firstheteropolymeric target sequence. The heteropolymeric probe sequence isbonded to the first heteropolymeric target sequence by Watson-Crickcomplementary base bonding and is also bonded to the secondheteropolymeric target sequence by homologous base bonding.

[0090] In certain embodiments, the probe and the target aredouble-stranded and the resulting complex is a quadruplex. This complexcan be free of PNA.

[0091] In certain embodiments, the quadruplex contains a heteropolymericprobe sequence parallel or antiparallel to a heteropolymeric targetsequence, wherein the heteropolymeric probe sequence is bonded to theheteropolymeric target sequence by homologous base binding orWatson-Crick complementary base binding. In such embodiments, thequadruplex complex contains a first probe strand containing saidheteropolymeric probe sequence and a second probe strand containing asecond heteropolymeric probe sequence that is complementary andantiparallel to the first probe sequence. The target includes a firsttarget strand containing a heteropolymeric target sequence and a secondtarget strand containing a second heteropolymeric target sequence thatis complementary and antiparallel to the first.

[0092] In such quadruplex embodiments, the heteropolymeric probesequence can bond to the heteropolymeric target sequence by Watson-Crickcomplementary or homologous base binding and the heteropolymeric probesequence can optionally and additionally bond to the secondheteropolymeric target sequence by homologous or Watson-Crickcomplementary base binding, respectively. Thus, when the heteropolymericprobe sequence bonds to the heteropolymeric target sequence byhomologous base bonding, the heteropolymeric probe sequence optionallybonds to the second heteropolymeric target sequence by Watson-Crickcomplementary base bonding, and when the heteropolymeric probe sequencebonds to the heteropolymeric target sequence by Watson-Crickcomplementary base bonding, the heteropolymeric probe sequenceoptionally bonds to the second heteropolymeric target sequence byhomologous base bonding.

[0093] In the purification method of the invention, probes are used topurify specific targets from samples by forming the above-describednovel complexes between the targets and probes. The complexes areseparated from the samples, and then the targets are separated from theprobes.

[0094] Thus, it is possible to purify double-stranded DNA target from asolution by contacting the target with a probe covalently bound to asupport, wherein the probe and target form a triplex, quadruplex or ahomologous duplex. This method is rapid without a prior denaturationstep, and can lead to high yields and degrees of purity with the removalof proteins, lipopolysaccharides, nucleases and the like. Moreover, themethod enables a nucleobase-containing target (e.g., dsDNA) to bepurified from complex mixtures of other nucleic acids, proteins,lipopolysaccharides and/or nucleases, and the like.

[0095] Referring to FIG. 15, which shows the coupling of single-strandedoligonucleotide probe 20 to target 22 (dsDNA), probe 20 is attached tostreptavidin-coated solid support 24 via biotin 26. Numerous types ofsolid supports are suitable for immobilizing oligonucleotides. Thesesolid supports are used in many formats such as membranes, microtiterplates, beads, probes, dipsticks, microwell, column, capillary tube,etc. A wide variety of chemical procedures are known to covalently linkoligonucleotides directly or through a linker to these solid supports.Of particular interest is the use of glass and nylon surfaces in thepreparation of DNA microarrays, which have been described in recentyears.

[0096] The following protocol details how an biotinylated oligo iscoupled to paramagnetic beads through a biotin/streptavidin linkage.

[0097] 1. Provide 1 ml streptavidin-conjugated paramagnetic beads (e.g.,Dynabeads M280 available from Dynal). The beads can be concentrated in amicrofuge tube using a magnet. The beads are superparamagnetic,polystyrene beads with streptavidin covalently attached to the beadsurface.

[0098] 2. Wash the beads three times with TE (Tris-EDTA) buffer and 0.5M NaCl by repeated concentration and resuspension.

[0099] 3. Quantify the biotinylated probe and add to the concentratedbeads.

[0100] 4. Mix and incubate for 30 minutes. Quantify the DNA in solutionafter concentration of the beads every 10 minutes by UV absorbance or byagarose gel. The amount of the probe that binds is related to thefraction of the probes that are biotinylated and the capacity of thebeads for the specific probe.

[0101] 5. The unbound DNA can be analyzed on an agarose gel +/−streptavidin or streptavidin IgG conjugate to determine if it isbiotinylated. The streptavidin will cause a mobility shift of thebiotinylated DNA. The excess biotinylated DNA can be incubated withfresh beads to generate more immobilized probe.

[0102] 6. Wash the beads three times with TE buffer and 0.5 M NaClfollowed by twice with TE buffer.

[0103] 7. Resuspend into 0.5 ml TE buffer with final concentration 150mg/ml. Use 10 μl for each capture reaction.

[0104] Store the beads at 4° C.

[0105] An oligonucleotide probe can be coupled to a column as follows.Streptavidin agarose (Molecular Probes, Eugene, Oreg., USA) is used asthe chromatographic matrix. The oligonucleotide probe is coupled throughits 5′-biotin terminal moiety to streptavidin groups on the agarosematrix.

[0106]1. Thoroughly resuspend the streptavidin agarose by inversion. Add1 ml of the slurry to a 2 ml chromatography column (Bio-Rad, Hercules,Calif., USA).

[0107] 2. Pre-equilibrate the column by washing the settled matrix with5×1 ml hybridization buffer (10 mM Tris HCl, pH 7.5, 15 mM MnCl₂ and 15mM MgCl₂) just before use.

[0108] Affinity plates can also be used to purify targets. Referring toFIGS. 16A-C, a sample containing target 30, biotinylated probe 32, andimpurities 34 and 36, is added to well 38, which is coated withstreptavidin 40. Target 30 (e.g., a plasmid as shown) specificallyassociates with biotinylated probe 32 to form a duplex, triplex orquadruplex of the invention. The probe-target complex is then attachedto well 38 through the interaction of the biotin of probe 32 andstreptavidin 40, as shown in FIG. 16B. Impurities 34 and 36 are thenwashed away, followed by the release of purified target 30, as shown inFIG. 16C.

[0109] Alternatively, well 38 can be coated with streptavidin 40 towhich biotinylated probe 32 is bound before the addition of sample tothe well. In this case, the sample, containing target 30 and impurities34 and 36, is added to well 38 to form a probe-target complex bonded towell 38 through the interaction of the biotin of probe 32 andstreptavidin 40, as shown in FIGS. 17A-17B. Impurities 34 and 36 arethen washed away, followed by the release of purified target 30, asshown in FIG. 17C.

[0110] An affinity plate for use in the foregoing method can be preparedaccording to the following preferred method:

[0111] 1. Wash the plate wells 6 times with TE buffer and 0.5 M NaCl byrepeated concentration and resuspension.

[0112] 2. Add the purified biotinylated probe to each well.

[0113] 3. Mix and incubate for 90 minutes. Quantify the DNA in solutionafter removal from the well by UV absorbance or by agarose gel. Theamount of the probe that binds is related to the fraction of the primersthat are biotinylated and the capacity of the well for the specificprobe.

[0114] 4. Wash the wells 6 times with TE buffer and 0.5 M NaCl followedby 4 times with TE buffer. Store the plates at 4° C.

[0115] 5. Pre-equilibrate the wells in hybridization buffer (10 mMTris-HCl, pH 7.5, 15 mM MnCl₂ and 15 mM MgCl₂) just before use.

[0116] Probes used in the purification method of the invention need notbe single-stranded. As shown in FIG. 18 double-stranded probes are alsowithin the scope of the invention. FIG. 18 shows the coupling ofdouble-stranded oligonucleotide probe 20 to target 22 (ssDNA), whereinprobe 20 is attached to streptavidin-coated solid support 24 via biotin26. FIG. 18 shows the coupling of a double-stranded oligonucleotideprobe 20 to ssDNA 22, probe 20 is attached to streptavidin-coated solidsupport 24 via biotin 26. Streptavidin tolerates a wide range ofconditions including 0.2M NaOH, 1% (w/v) SDS and prolonged heating at70° C. Heating to 90° C. gradually decreases activity. One biotinylatedprobe (2.5 to 5 pmole per 50 μl) is employed to capture the targetnucleic acid at the correct annealing temperature during a 30-minuteincubation time. After a rapid wash step, the captured nucleic acids canbe isolated.

[0117] Capture speed and/or efficiency can be enhanced in certainembodiments by employing the dynamic hybridization technology describedin U.S. Pat. No. 6,255,050 to Nie et al. In such embodiments, thecapture apparatus includes means for moving the target into proximitywith the probe by moving the sample over or past the support, to whichthe probe is bound, at least once. For example, the sample can bealternately pushed and pulled through a membrane on which the probe isbound.

[0118] The invention will be illustrated in more detail with referenceto the following Examples, but it should be understood that the presentinvention is not deemed to be limited thereto.

EXAMPLES Example 1

[0119] Complementary sense and antisense 50-mer ssDNA target sequences,derived from exon 10 of the human cystic fibrosis gene (Nature 380, 207(1996)) were synthesized on a DNA synthesizer (Expedite 8909, PerSeptiveBiosystems) and purified by HPLC. SsDNA oligonucleotides were dissolvedin ddH₂O and diluted to a concentration of 1 pmole/μl. Equimolar amountsof complementary oligonucleotides were heated at 95° C. for 10 min andallowed to anneal gradually in the presence of 10 mM Tris, pH 7.5, 1 mMEDTA and 100 mM NaCl, as the temperature cooled to 21° C. over 1.5hours. DsDNA oligonucleotides were diluted in ddH₂O at a concentrationof 1 pmole/μl.

[0120] The sequence for the sense strand of the wild-type target DNA(SEQ ID NO:1) was: 5′-TGG CAC CAT TAA AGA AAA TAT CAT CTT TGG TGT TTCCTA TGA TGA ATA TA-3′.

[0121] The sequence for the antisense strand of the wild-type target DNA(SEQ ID NO:1) was: 5′-TAT ATT CAT CAT AGG AAA CAC CAA AGA TGA TAT TTTCTT TAA TGG TGC CA-3′.

[0122] SEQ ID NO:2 was a 50-mer mutant dsDNA target sequence identicalto wild-type target DNA (SEQ ID NO:1) except for a one base pairmutation (underlined) at amino acid position 507 at which the wild-typesense strand sequence CAT was changed to CGT.

[0123] The sequence for the sense strand of SEQ ID NO:2 was: 5′-TGG CACCAT TAA AGA AAA TAT CGT CTT TGG TGT TTC CTA TGA TGA ATA TA-3′.

[0124] The sequence for the antisense strand of SEQ ID NO:2 was: 5′-TATATT CAT CAT AGG AAA CAC CAA AGA C GA TAT TTT CTT TAA TGG TGC CA-3′.

[0125] SEQ ID NO:3 was a 50-mer mutant dsDNA target sequence identicalto wild-type target DNA (SEQ ID NO:1) except for a consecutive two basepair mutation (underlined) at amino acid positions 506 and 507 at whichthe wild-type sense strand sequence CAT was changed to ACT.

[0126] The sequence for the sense strand of SEQ ID NO:3 was: 5′-TGG CACCAT TAA AGA AAA TAT ACT CTT TGG TGT TTC CTA TGA TGA ATA TA-3′.

[0127] The sequence for the antisense strand of SEQ ID NO: 3 was: 5′-TATATT CAT CAT AGG AAA CAC CAA AGA GT A TAT TTT CTT TAA TGG TGC CA-3′.

[0128] SEQ ID NO:4 was a 50-mer mutant dsDNA target sequence identicalto wild-type target DNA (SEQ ID NO:1) except for a consecutive threebase pair mutation (underlined) at amino acid positions 506 and 507 atwhich the wild-type sense strand sequence CAT was changed to ACG.

[0129] The sequence for the sense strand of SEQ ID NO:4 was: 5′-TGG CACCAT TAA AGA AAA TAT ACG CTT TGG TGT TTC CTA TGA TGA ATA TA-3′.

[0130] The sequence for the antisense strand of SEQ ID NO:4 was: 5′-TATATT CAT CAT AGG AAA CAC CAA ACG GTA TAT TTT CTT TAA TGG TGC CA-3′.

[0131] SEQ ID NO:5 was a 50-mer dsDNA target sequence modified from SEQID NO:1, wherein the percent GC content was changed from 30% to 52%.

[0132] The sequence for the sense strand of the wild-type target DNA(SEQ ID NO:5) was: 5′-GAG CAC CAT GAC AGA CAC TGT CAT CTC TGG TGT GTCCTA CGA TGA CTC TG-3′.

[0133] The sequence for the antisense strand of the wild-type target DNA(SEQ ID NO:5) was: 5′-CAG AGT CAT CGT AGG ACA CAC CAG AGA TGA CAG TGTCTG TCA TGG TGC TC-3′.

[0134] SEQ ID NO:6 was a 50-mer mutant dsDNA target sequence identicalto SEQ ID NO:5, except for a one base pair mutation (underlined), atwhich the sense strand sequence CAT was changed to CGT.

[0135] The sequence for the sense strand of mutant SEQ ID NO:6 was:5′-GAG CAC CAT GAC AGA CAC TGT CGT CTC TGG TGT GTC CTA CGA TGA CTCTG-3′.

[0136] The sequence for the antisense strand of mutant SEQ ID NO:6 was:5′-CAG AGT CAT CGT AGG ACA CAC CAG AGA CGA CAG TGT CTG TCA TGG TGCTC-3′.

[0137] SEQ ID NO:7 was a 50-mer mutant dsDNA target sequence identicalto SEQ ID NO:5, except for a one base pair mutation (underlined), atwhich the sense strand sequence CTC was changed to CTT.

[0138] The sequence for the sense strand of mutant SEQ ID NO:7 was:5′-GAG CAC CAT GAC AGA CAC TGT CAT CTT TGG TGT GTC CTA CGA TGA CTCTG-3′.

[0139] The sequence for the antisense strand of mutant SEQ ID NO:7 was:5′-CAG AGT CAT CGT AGG ACA CAC CAA AGA TGA CAG TGT CTG TCA TGG TGCTC-3′.

[0140] SEQ ID NO:8 was a 50-mer mutant dsDNA target sequence identicalto SEQ ID NO:5, except for a consecutive two base pair mutation(underlined), at which the sense strand sequence CAT was changed to ACT.

[0141] The sequence for the sense strand of mutant SEQ ID NO:8 was:5′-GAG CAC CAT GAC AGA CAC TGT ACT CTC TGG TGT GTC CTA CGA TGA CTCTG-3′.

[0142] The sequence for the antisense strand of mutant SEQ ID NO:8 was:5′-CAG AGT CAT CGT AGG ACA CAC CAG AGA GTA CAG TGT CTG TCA TGG TGCTC-3′.

[0143] SEQ ID NO:9 was a 47-mer mutant dsDNA target sequence identicalto wild-type target DNA (SEQ ID NO: 1) except for a consecutive threebase pair deletion (indicated by three dots) at amino acid positions 507and 508 at which the wild-type sense strand sequence CTT is deleted.

[0144] The sequence for the sense strand of SEQ ID NO:9 was: 5′-TGG CACCAT TAA AGA AAA TAT CAT . . . TGG TGT TTC CTA TGA TGA ATA TA-3′.

[0145] The sequence for the antisense strand of SEQ ID NO:9 was: 5′-TATATT CAT CAT AGG AAA CAC CA . . . A TGA TAT TTT CTT TAA TGG TGC CA-3′.

[0146] SEQ ID NO:10 was a 50-mer mutant dsDNA target sequence identicalto SEQ ID NO:5, except for a one base pair mutation (underlined), atwhich the sense strand sequence CAT was changed to CTT.

[0147] The sequence for the sense strand of mutant SEQ ID NO:10 was:5′-GAG CAC CAT GAC AGA CAC TGT CTT CTC TGG TGT GTC CTA CGA TGA CTCTG-3′.

[0148] The sequence for the antisense strand of mutant SEQ ID NO:10 was:5′-CAG AGT CAT CGT AGG ACA CAC CAG AGA AGA CAG TGT CTG TCA TGG TGCTC-3′.

[0149] SEQ ID NO:11 was a 50-mer mutant dsDNA target sequence identicalto SEQ ID NO: 5, except for a one base pair mutation (underlined), atwhich the sense strand sequence CTC was changed to CCC.

[0150] The sequence for the sense strand of mutant SEQ ID NO:11 was:5′-GAG CAC CAT GAC AGA CAC TGT CAT CCC TGG TGT GTC CTA CGA TGA CTCTG-3′.

[0151] The sequence for the antisense strand of mutant SEQ ID NO:11 was:5′-CAG AGT CAT CGT AGG ACA CAC CAG GGA TGA CAG TGT CTG TCA TGG TGCTC-3′.

[0152] The PNA probes were synthesized, HPLC purified and confirmed bymass spectroscopy by Commonwealth Biotechnologies, Inc. (Richmond, Va.,USA). PNA probes were first dissolved in 0.1% TFA (trifluoroacetic acid)to a concentration of 10 mg/ml, and then diluted to 1 mg/ml by theaddition of ddH₂O. Final PNA stock solutions were prepared in ddH₂O at aconcentration of 1 pmole/μl.

[0153] Probe No. 1 was a 15-mer PNA probe designed to be completelycomplementary to a 15 nucleotide segment of the sense strand of the50-mer wild-type target DNA (SEQ ID NO:1), overlapping amino acidpositions 505 to 510 (Nature 380, 207 (1996)). The directionality of theprobe was opposite or antiparallel to that of the sense strand in thetarget.

[0154] The sequence for Probe No. 1 (SEQ ID NO:12) was: 5′-H-CAC CAA AGATGA TAT-Lys-CONH₂-3′.

[0155] Probe No. 2 was a 15-mer PNA probe identical in sequence to ProbeNo. 1, but was of the same directionality, or parallel to that of thesense strand in the dsDNA target.

[0156] The sequence for Probe No. 2 (SEQ ID NO:13) was: 5′-H-TAT AGT AGAAAC CAC-Lys-CONH₂-3′.

[0157] The 15-mer ssDNA probes were synthesized and purified by HPLC asabove. SsDNA probes were dissolved in ddH₂O at a concentration of 1pmole/μl.

[0158] Probe No. 3 was a 15-mer ssDNA probe designed to be completelycomplementary to a 15 nucleotide segment of the sense strand of the50-mer wild-type target DNA (SEQ ID NO:5). The directionality of theprobe was opposite or antiparallel to that of the sense strand in thetarget.

[0159] The sequence for Probe No. 3 (SEQ ID NO:14) was: 5′-CAC CAG AGATGA CAG-3′.

[0160] Probe No. 4 was a 15-mer ssDNA probe identical in sequence toProbe No. 3, but was of the same directionality, or parallel to that ofthe sense strand in the dsDNA target.

[0161] The sequence for Probe No. 4 (SEQ ID NO:15) was: 5′-GAC AGT AGAGAC CAC-3′.

[0162] Probe No. 5 was a 15-mer antiparallel ssDNA probe identical toProbe No. 3, except it had an attached fluorescein moiety at the 5′position.

[0163] The sequence for Probe No. 5 (SEQ ID NO:16) was: 5′-Flu-CAC CAGAGA TGA CAG-3′.

[0164] Probe No. 6 was a 15-mer parallel ssDNA probe identical to ProbeNo. 4, except it had an attached fluorescein moiety at the 5′ position.

[0165] The sequence for Probe No. 6 (SEQ ID NO:17) was: 5′-Flu-GAC AGTAGA GAC CAC-3′.

[0166] Probe No. 7 was a 15-mer ssDNA probe, with an attachedfluorescein moiety at the 5′ position, designed to be completelycomplementary to a 15 nucleotide segment of the sense strand of the50-mer wild-type target DNA (SEQ ID NO:1). The directionality of theprobe was opposite or antiparallel to that of the sense strand in thetarget.

[0167] The sequence for Probe No. 7 (SEQ ID NO:18) was: 5′-Flu-CAC CAAAGA TGA TAT-3′.

[0168] Probe No. 8 was a 15-mer ssDNA probe designed to be completelycomplementary to a 15 nucleotide segment of the sense strand of the50-mer wild-type target DNA (SEQ ID NO:1). The directionality of theprobe was antiparallel to that of the sense strand in the target.

[0169] The sequence for Probe No. 8 (SEQ ID NO:19) was: 5′-CAC CAA AGATGA TAT-3′.

[0170] Probe No. 9 and Probe No. 10 were 15-mer mutant ssDNA probesidentical in sequence to wild-type Probe No. 8, except for a one basemutation (underlined).

[0171] The sequence for Probe No. 9 (SEQ ID NO:20) was: 5′-CAC GAA AGATGA TAT-3′.

[0172] The sequence for Probe No. 10 (SEQ ID NO:21) was: 5′-CAC CAA ACATGA TAT-3′.

[0173] It is well known that ssDNA strands of mixed base sequencereadily form ssPNA:ssDNA duplexes on a Watson-Crick pairing basis whenreacted with either antiparallel or parallel synthesized ssPNA strandsat room temperature. We have previously shown that such ssPNA:ssDNAcomplexes containing perfectly matched sequences can reliably bedistinguished from ssPNA:ssDNA complexes containing a 1 bp mismatch whenassayed in the presence of the DNA intercalator, YOYO-1 (MolecularProbes, Eugene, Oreg., USA), and that the order of assembly of the PNAstrand has a significant bearing on its ability to specifically bind assDNA target. Example 1 compares the efficiency of formation of dsDNAduplexes when wild-type or mutant ssDNA target sequences are reactedwith Watson-Crick complementary antiparallel ssDNA probes or withhomologous, that is to say identical parallel, ssDNA probes.

[0174] The hybridization reaction mixtures giving rise to the dataillustrated in FIG. 1A, each contained the following mixture: 2 pmolesof ssDNA target, 2 pmoles of ssDNA probe, 0.5×TBE and 500 nM of YOYO-1in a final volume of 40 μl. The reaction mixtures were incubated at roomtemperature (21° C.) for 5 minutes, placed into a quartz cuvette,irradiated with an argon ion laser beam having a wavelength of 488 nmand monitored for fluorescent emission. The intensity of fluorescencewas plotted as a function of wavelength for each sample analyzed.

[0175] In FIGS. 1B and 1C, the hybridization reaction mixtures (40 μl)each contained the following: 2 pmoles of ssDNA target, 2 pmoles of5′-fluorescein labeled ssDNA probe, 10 mM Tris-HCl, pH 7.5, and 1 mMEDTA. The reaction mixtures were incubated at room temperature (21° C.)for 30 minutes or 90 minutes. Following incubation, each sample wasplaced into a quartz cuvette, irradiated with an argon ion laser beamhaving a wavelength of 488 nm and monitored for fluorescent emission.The maximum fluorescent intensities occurred at a wavelength of 525 nm,the emission wavelength for fluorescein. The intensity of fluorescentemission was plotted as a function of wavelength for each sampleanalyzed.

[0176] When the ssDNA Probe No. 3 was reacted with the 50-mer wild-typesense strand of SEQ ID NO:5 or with the 50-mer mutant sense strand ofSEQ ID NO:7 in the presence of YOYO-1, antiparallel complementaryssDNA:ssDNA duplexes were formed (FIG. 1A). The fluorescent intensityemitted by the 1 bp T-G mismatched antiparallel complementary duplex(sense strand of SEQ ID NO:7+Probe No. 3) was 56% lower than thatobtained by the perfectly matched antiparallel complementary duplex(sense strand of SEQ ID NO:5+Probe No. 3).

[0177] When the ssDNA Probe No. 3 was reacted with the 50-mer wild-typeantisense strand of SEQ ID NO:5 in the presence of YOYO-1, theefficiency of parallel homologous ssDNA:ssDNA duplex formation was only3% lower than the efficiency of antiparallel complementary ssDNA:ssDNAduplex formation (FIG. 1A). This result was completely unanticipated.The 1 bp A-G mismatched parallel homologous duplex formed when the50-mer mutant antisense strand of SEQ ID NO:7 was reacted with the ssDNAProbe No. 3 in the presence of YOYO-1, produced a fluorescent emissionintensity that was 56% lower than that emitted by the perfectly parallelhomologous duplex (FIG. 1A). Control samples comprising each 50-merssDNA target plus 500 nM YOYO-1 exhibited levels of fluorescence whichranged from 91% to 92% lower than that observed with the perfectlymatched duplexes (FIG. 1A). The level of fluorescence emitted by the15-mer ssDNA Probe No. 3 plus 500 nM YOYO-1 was slightly greater thanthat produced by YOYO-1 alone. The shift in fluorescent emissionwavelength observed with the ssDNA targets and probe is typical ofYOYO-1's emission profile in the presence of ssDNA.

[0178] YOYO-1 facilitated DNA complex formation between a ssDNA probeand a complementary base sequence in an antiparallel ssDNA target, orbetween a ssDNA probe and an identical base sequence in a parallel ssDNAtarget, with similar efficacy, to allow differentiation betweenperfectly matched complexes and those containing a 1 bp mismatch. In theparallel homologous complexes, the 1 bp mismatch was a non-homologousbase pair.

[0179] The comparative efficiency of antiparallel complementary andparallel homologous dsDNA duplex formation was further examined usingssDNA targets and ssDNA-F probes in the absence of complex promotingagents such as YOYO-1 or cations. When the ssDNA-F Probe No. 5 wasincubated for 30 minutes in Tris buffer at room temperature with the50-mer wild-type sense strand of SEQ ID NO:5, the Watson-Crickcomplementary antiparallel ssDNA:ssDNA-F duplexes were formed veryefficiently, resulting in a 53% reduction in fluorescent emissioncompared to that emitted by Probe No. 5 alone (FIG. 1B). By contrast,antiparallel complementary ssDNA:ssDNA-F complexes that contained a 1 bpT-G mismatch (sense strand of SEQ ID NO:7+Probe No. 5) were less stable,resulting in only a 40% decrease in fluorescent emission compared tothat emitted by Probe No. 5 alone after a 30 minute incubation (FIG.1B).

[0180] Parallel homologous ssDNA:ssDNA-F complexes were formed when thessDNA Probe No. 5 was reacted with the 50-mer wild-type antisense strandof SEQ ID NO:5 or with the 50-mer mutant antisense strand of SEQ IDNO:7, generating fluorescent emission intensities that were 44% and 37%lower, respectively, than that emitted by ssDNA Probe No. 5 alone aftera 30 minute incubation (FIG. 1B). The avid formation of parallelhomologous ssDNA:ssDNA-F complexes in the absence of a promoting agentwas completely unanticipated. The discrimination between signals emittedfrom perfectly matched duplexes and 1 bp mismatched duplexes in theabsence of complex promoting agents, was not as dramatic as thatobserved when YOYO-1 was present and served as the promoter andsignaling agent (compare FIGS. 1A and 1B). This was the case for bothantiparallel and parallel duplexes. Slightly less discrimination betweenperfectly matched and 1 bp mismatched DNA complexes was observed when aparallel homologous ssDNA target was used than when an antiparallelcomplementary ssDNA target was used to produce the ssDNA:ssDNA-Fcomplexes (FIG. 1B).

[0181] After a 90 minute incubation, Watson-Crick antiparalleldsDNA:ssDNA-F complexes consisting of perfectly complementary sequences(sense strand of SEQ ID NO:5+Probe No. 5) or 1 bp T-G mismatchedsequences (sense strand of SEQ ID NO:7+Probe No. 5) produced a 39% and30% decrease, respectively, in fluorescent emission intensity comparedto that emitted by Probe No. 5 alone (FIG. 1C). Remarkably, parallelhomologous ssDNA:ssDNA-F complexes exhibited the same level of stabilityafter 90 minutes of incubation as did the Watson-Crick antiparallelssDNA:ssDNA-F complexes. The fluorescent intensities for a perfectlyparallel homologous duplex (antisense strand of SEQ ID NO:5+Probe No. 5)and a 1 bp A-G mismatched parallel homologous duplex (antisense strandof SEQ ID NO:7+Probe No. 5) were 40% and 25% lower, respectively, thanthat emitted by ssDNA Probe No. 5 alone after a 90 minute incubation(FIG. 1C).

[0182] The mechanism of recognition and binding of the homologous basesin the parallel dsDNA duplexes is unknown at this time. Nevertheless,recognition and binding of parallel homologous ssDNA sequences occurredin a configuration which allowed the discrimination between perfectlymatched ssDNA:ssDNA complexes and those containing a 1 bp or 2 bpmismatch. In these parallel homologous complexes, the 1 bp mismatch wasa non-homologous base pair.

Example 2

[0183] In Example 1, the remarkable efficiency of parallel homologousssDNA:ssDNA duplex formation was demonstrated both in the presence of acomplex promoting agent such as YOYO-1 and in the absence of any complexpromoting agent. The recognition and binding of the homologous bases inthe parallel dsDNA duplexes was such as to allow easy discriminationbetween perfectly homologous base sequences and parallel homologoussequences that contained a 1 bp mismatch. These parallel homologous 1 bpmismatches were also clearly recognizable as mismatches based onWatson-Crick complementary recognition and binding rules. Example 2examines the recognition and binding efficiency of parallel homologousdsDNA duplexes that contain A-T or G-C base pairings, to determinewhether these Watson-Crick complementary pairings appear as mismatchesin a parallel homologous binding reaction.

[0184] Each hybridization reaction mixture (40 μl) contained thefollowing: 2 pmoles of ssDNA target, 2 pmoles of ssDNA probe, 0.5×TBEand 500 nM of YOYO-1. The reaction mixtures were incubated at roomtemperature (21° C.) for 5 minutes, placed into a quartz cuvette,irradiated with an argon ion laser beam having a wavelength of 488 nmand monitored for fluorescent emission. The intensity of fluorescencewas plotted as a function of wavelength for each sample analyzed.

[0185] When the ssDNA Probe No. 3 (with a 53% GC content) was reactedwith the 50-mer wild-type antisense strand of SEQ ID NO:5 or with the50-mer mutant antisense strand of SEQ ID NO:10 in the presence ofYOYO-1, parallel homologous ssDNA:ssDNA duplexes were formed (FIG. 2A).The fluorescent intensity emitted by the 1 bp A-T mismatched parallelhomologous duplex (antisense strand of SEQ ID NO:10+Probe No. 3) was 72%lower than that obtained by the perfectly parallel homologous duplex(antisense strand of SEQ ID NO:5+Probe No. 3) (FIG. 2A). This dramaticdecrease in fluorescent emission by the parallel homologous duplexcontaining a 1 bp A-T, strongly suggested that the Watson-Crick A-Tbinding was hindered by the spatial and/or charge configuration imposedon the A and T bases when part of parallel homologous strands attemptingto achieve stable duplex. Control samples comprising each 50-mer ssDNAtarget plus 500 nM YOYO-1 exhibited levels of fluorescence which rangedfrom 96% to 97% lower than that observed with the perfectly matchedduplexes (FIG. 2A). The level of fluorescence emitted by the 15-merssDNA Probe No. 3 plus 500 nM YOYO-1 was slightly greater than thatproduced by YOYO-1 alone. The shift in fluorescent emission wavelengthobserved with the ssDNA targets and probe is typical of YOYO-1'semission profile in the presence of ssDNA.,

[0186] Parallel homologous ssDNA:ssDNA duplexes were also formed whenthe 50-mer wild-type antisense strand of SEQ ID NO:1 (with a 33% GCcontent) was reacted with the wild-type ssDNA Probe No. 8 or with themutant ssDNA Probes No. 9 and 10, in the presence of YOYO-1 (FIG. 2B).The fluorescent intensities emitted by the 1 bp G-C mismatched parallelhomologous duplex (antisense strand of SEQ ID NO:1+Probe No. 9) and the1 bp C-G mismatched parallel homologous duplex (antisense strand of SEQID NO:1+Probe No. 10) were 67% and 66% lower, respectively, than thatobtained by the perfectly parallel homologous duplex (antisense strandof SEQ ID NO:1+Probe No. 8) (FIG. 2B). The configuration of theinteracting bases in the parallel homologous duplexes was unfavorablefor Watson-Crick complementary G-C binding, resulting in a decrease influorescent emission indicative of a 1 bp mismatch. Control samplesconsisting of the 50-mer ssDNA target plus 500 nM YOYO-1 or each of the15-mer ssDNA probes plus 500 nM YOYO-1 resulted in levels offluorescence that were slightly greater than that produced by YOYO-1alone (FIG. 2B).

[0187] Therefore, the interacting base pairs in parallel homologousdsDNA duplexes, formed in the presence of YOYO-1, adopt a configurationthat is unfavorable for binding between Watson-Crick complementary basepairs, resulting in such duplexes appearing to contain 1 bp mismatches.

[0188] We are led to envisage how mismatches in binding sequences,whether occurring as part of a hairpin or multistrand complex can causeenergetic and repeated motion as the base sequences try to achieve thestability of the ideal binding configuration under either binding motif.It is expected that binding strength of base pairs upstream ordownstream of nucleation sites, metal ions and other factors will have abearing on the attempts to achieve bonding.

Example 3

[0189] This example examines the efficiency of antiparallel homologousssDNA:ssDNA duplex formation facilitated by YOYO-1 or by monovalentcations.

[0190] The hybridization reactions, giving rise to the data illustratedin FIG. 3A, each contained the following mixture: 2 pmoles of ssDNAtarget, 2 pmoles of ssDNA probe, 0.5×TBE and 500 nM of YOYO-1 in a finalvolume of 40 μl. The reaction mixtures were incubated at roomtemperature (21° C.) for 5 minutes, placed into a quartz cuvette,irradiated with an argon ion laser beam having a wavelength of 488 nmand monitored for fluorescent emission. The intensity of fluorescentemission was plotted as a function of wavelength for each sampleanalyzed.

[0191] In FIG. 3B, the hybridization reaction mixtures (40 82 l) eachcontained the following: 2 pmoles of ssDNA target, 2 pmoles of5′-fluorescein labeled ssDNA probe, 10 mM Tris-HCl, pH 7.5, and 50 mMNaCl. The reaction mixtures were incubated at room temperature (21° C.)for various lengths of time ranging from 1 minute to 60 minutes.Following incubation, samples were placed into a quartz cuvette,irradiated with an argon ion laser beam having a wavelength of 488 nmand monitored for fluorescent emission. The intensity of fluorescentemission was plotted as a function of wavelength for each sampleanalyzed.

[0192] Incubation of ssDNA Probe No. 4 with the 50-mer wild-typeantisense strand of SEQ ID NO:5 in the presence of YOYO-1 resulted inantiparallel homologous ssDNA:ssDNA complex formation (FIG. 3A).Although the efficiency of antiparallel homologous complex formation wasonly 65% that of conventional antiparallel complementary dsDNA formation(compare FIGS. 1A and 3A), recognition and binding of antiparallelhomologous ssDNA sequences did occur, facilitated by YOYO-1. This resultwas completely unanticipated. Furthermore, antiparallel homologousssDNA:ssDNA complexes comprising wild-type sequences were clearlydistinguished from those comprising 1 bp or 2 bp mismatches. Thefluorescent intensities emitted by the 1 bp A-G mismatched DNA complex(antisense strand of SEQ ID NO:7+Probe No. 4), the 1 bp C-T mismatchedDNA complex (antisense strand of SEQ ID NO:6+Probe No. 4), and theconsecutive 2 bp mismatched DNA complex (antisense strand of SEQ IDNO:8+Probe No. 4) were 25%, 65% and 71% lower, respectively, than thatobtained by the perfect antiparallel homologous complex (antisensestrand of SEQ ID NO:5+Probe No. 4) (FIG. 3A). As the degree of homologybetween the probe and target decreased, the level of fluorescentemission decreased. Control samples comprising each 50-mer ssDNA targetplus 500 nM YOYO-1 exhibited levels of fluorescence which ranged from88% to 90% lower than that observed with the perfectly matched complexes(FIG. 3A). The level of fluorescence emitted by the 15-mer ssDNA ProbeNo. 4 plus 500 nM YOYO-1 was slightly greater than that produced byYOYO-1 alone.

[0193] Antiparallel homologous ssDNA:ssDNA complex formation was furtherexamined using ssDNA targets and ssDNA-F probes both in the presence andabsence of 50 mM NaCl. After 15 minutes of incubation of ssDNA-F ProbeNo. 6 with the 50-mer wild-type antisense strand of SEQ ID NO:5 in thepresence of 50 mM NaCl, antiparallel homologous ssDNA:ssDNA-F complexeswere formed, as indicated by the 34% decrease in fluorescence observedcompared to that emitted by Probe No. 6 alone (FIG. 3B). The efficiencyof antiparallel homologous complex formation was 62% that ofantiparallel complementary complex formation following a 15 minuteincubation (data not shown). By contrast, antiparallel homologousssDNA:ssDNA-F complexes that contained a 1 bp A-G mismatch (antisensestrand of SEQ ID NO:7+Probe No. 6), a 1 bp C-T mismatch (antisensestrand of SEQ ID NO:6+Probe No. 6), a 1 bp A-T mismatch (antisensestrand of SEQ ID NO:10+Probe No. 6), and a consecutive 2 bp mismatch(antisense strand of SEQ ID NO:8+Probe No. 6), produced a 24%, 26%, 23%and a 13% decrease in fluorescence, respectively, compared to thatemitted by Probe No. 6 alone after a 15 minute incubation (FIG. 3B). Theconfiguration of the interacting bases in the antiparallel homologousduplexes was apparently unfavorable for Watson-Crick complementary A-Tbinding, resulting in a change in fluorescent emission indicative of a 1bp mismatch. Less antiparallel homologous complex formation occurredfollowing a 30 minute incubation in the presence of 50 mM NaCl (data notshown). No complex formation was evident after 45 minutes of incubation.Similar rates of antiparallel homologous complex formation and stabilitywere observed in Tris buffer without NaCl (data not shown).

[0194] Promoted by YOYO-1 or NaCl, recognition and binding ofantiparallel homologous ssDNA sequences occurred in a configurationwhich allowed the discrimination between perfectly matched ssDNA:ssDNAcomplexes and those containing a 1 bp or 2 bp mismatch. The interactionof the base pairs in the antiparallel homologous duplex resulted in aconventional Watson-Crick A-T base pair being destabilizing as amismatch.

Example 4

[0195] This example demonstrates the efficiency of parallelcomplementary ssDNA:ssDNA complex formation promoted by monovalentcations. The hybridization reaction mixtures (40 μl) each contained thefollowing: 2 pmoles of ssDNA target, 2 pmoles of 5′-fluorescein labeledssDNA probe, 10 mM Tris-HCl, pH 7.5, and 50 mM NaCl. The reactionmixtures were incubated at room temperature (21° C.) for various lengthsof time ranging from 1 minute to 60 minutes. Following incubation,samples were placed into a quartz cuvette, irradiated with an argon ionlaser beam having a wavelength of 488 nm and monitored for fluorescentemission. The intensity of fluorescent emission was plotted as afunction of wavelength for each sample analyzed.

[0196] After a 15 minute incubation in the presence of 50 mM NaCl,ssDNA:ssDNA-F duplexes consisting of perfectly complementary sequences(sense strand of SEQ ID NO:5+Probe No. 6) formed readily, resulting in a41% decrease in fluorescent emission intensity compared to that emittedby Probe No. 6 alone (FIG. 4). This high efficiency of parallelcomplementary duplex formation was completely unexpected. By contrast,incompletely complementary ssDNA:ssDNA-F complexes containing a 1 bp T-Gmismatch (sense strand of SEQ ID NO:7+Probe No. 6), a 1 bp G-T mismatch(sense strand of SEQ ID NO:6+Probe No. 6), a 1 bp T-T mismatch (sensestrand of SEQ ID NO:10+Probe No. 6), and a consecutive 2 bp mismatch(sense strand of SEQ ID NO:8+Probe No. 6), generated an 18%, 20%, 10%and 16% decrease, respectively, in fluorescent emission intensitycompared to that exhibited by Probe No. 6 alone (FIG. 4).

[0197] Once formed in the presence of 50 mM NaCl, the perfectly matchedparallel complementary duplexes were very stable, resulting in a 40% and47% decrease in fluorescent emission after 30 minutes and 45 minutes ofincubation, respectively, compared to that emitted by Probe No. 6 alone(data not shown). The 1 bp and 2 bp mismatched parallel complementarycomplexes were much less stable after 30 minutes and 45 minutes ofincubation in the presence of 50 mM NaCl (data not shown). The rate andefficiency of parallel complementary ssDNA:ssDNA-F formation was verysimilar to that of antiparallel complementary ssDNA:ssDNA-F formationduring the first 45 minutes of incubation in the presence of 50 mM NaCl(data not shown). While antiparallel complementary complexes continuedto form easily after 60 minutes of incubation in 50 mM NaCl, no parallelcomplementary complex formation was evident at this time (data notshown).

[0198] NaCl facilitated DNA complex formation between a ssDNA-F probeand an antiparallel complementary ssDNA target, or between a ssDNA-Fprobe and a parallel complementary ssDNA target, with similar efficacy,to allow differentiation between perfectly matched complexes and thosecontaining a 1 bp or 2 bp mismatch.

Example 5

[0199] Examples 1 to 4 demonstrated alternate base recognition andbinding motifs occurring between antiparallel or parallel ssDNA probes,and complementary or homologous ssDNA targets to generate ssDNA:ssDNAduplexes, other than the conventional antiparallel Watson-Crickcomplementary dsDNA complexes. This example will show that bases arecapable of recognizing and interacting with both complementary andhomologous bases at the same time.

[0200] Samples of two pmoles of ssDNA Probe No. 3 were heated at 95° C.for 10 minutes and allowed to cool to room temperature for 30 minutes inthe presence of various concentrations of a free base, resulting inssDNA probes containing conjugated bases. Duplicate samples of ssDNAProbe No. 3 were similarly denatured and cooled in the absence of addedfree bases to generate non-conjugated ssDNA probes. Two pmoles of theseconjugated or non-conjugated ssDNA probes were then mixed with 2 pmolesof ssDNA target in the presence of 500 nM YOYO-1 and 0.5×TBE in a finalreaction volume of 40 μl. The reaction mixtures were incubated at roomtemperature (21° C.) for 5 minutes, placed into a quartz cuvette,irradiated with an argon ion laser beam having a wavelength of 488 nm,and monitored for fluorescent emission. The intensity of fluorescencewas plotted as a function of wavelength for each sample analyzed.

[0201] When the non-conjugated ssDNA Probe No. 3 was reacted with the50-mer wild-type sense strand of SEQ ID NO:5 or with the 50-mer mutantsense strand of SEQ ID NO:7, in the presence of YOYO-1, antiparallelcomplementary ssDNA:ssDNA complexes were formed (FIG. 5A). Thefluorescent intensity emitted by the 1 bp T-G mismatched antiparallelcomplementary duplex (sense strand of SEQ ID NO:7+Probe No. 3) was 45%lower than that obtained by the perfectly matched antiparallelcomplementary duplex (sense strand of SEQ ID NO:5+Probe No. 3). Controlsamples comprising each 50-mer ssDNA target plus 500 nM YOYO-1 exhibitedlevels of fluorescence which ranged from 92% to 93% lower than thatobserved with the perfectly matched duplexes (FIG. 5A). The level offluorescence emitted by the 15-mer ssDNA Probe No. 3 plus 500 nM YOYO-1was slightly greater than that produced by YOYO-1 alone.

[0202] When the ssDNA Probe No. 3 was reacted with the 50-mer wild-typeantisense strand of SEQ ID NO:5 in the presence of YOYO-1, theefficiency of parallel homologous ssDNA:ssDNA duplex formation was 14%lower than the efficiency of antiparallel complementary ssDNA:ssDNAduplex formation (compare FIGS. 5A and 5B). The 1 bp A-G mismatchedparallel homologous duplex formed when the 50-mer mutant antisensestrand of SEQ ID NO:7 was reacted with the ssDNA Probe No. 3 in thepresence of YOYO-1, produced a fluorescent emission intensity that was47% lower than that emitted by the perfectly parallel homologous duplex(FIG. 5B).

[0203] The 15-mer ssDNA Probe No. 3 contains six adenine bases.Conjugation of 2 pmoles of ssDNA Probe No. 3 with 3 pmoles of freethymine could result in 25% of the complementary A or 100% of thehomologous T within Probe No. 3 bound to the added thymine.Complementary A-T binding is energetically preferred. Reaction of 2pmoles of ssDNA Probe No. 3 (conjugated with 3 pmoles of thymine) with 2pmoles of the wild-type antisense strand of SEQ ID NO:5 in the presenceof YOYO-1 resulted in dramatically enhanced parallel homologousssDNA:ssDNA complex formation (FIG. 5B). Twenty-five percent conjugationof the ssDNA probe with 3 pmoles of thymine increased parallelhomologous complex formation between the perfectly homologous sequencesby 78%. This augmentation of parallel homologous complex formation canbe linked to the ability of the adenines in Probe No. 3 to interactsimultaneously with the conjugated complementary thymine bases, as wellas with the homologous adenines in the ssDNA target. Moreover,interaction with available complementary bases was not deleterious tothe homologous binding configuration adopted by the homologous bases andtheir neighbors.

[0204] By contrast, the efficiency of formation of parallel homologouscomplexes containing a 1 bp A-G mismatch (antisense strand of SEQ IDNO:7+Probe No. 3) were increased by 16% when Probe No. 3 was conjugated25% with thymine than when non-conjugated Probe No. 3 was used (FIG.5B). This corresponded to a 65% reduction in fluorescent emissionintensity for the 1 bp A-G mismatched parallel homologous complexcompared to that observed for the perfectly matched parallel homologouscomplex when the T-conjugated Probe No. 3 was used. Conjugation of thessDNA probe increased the specificity in discriminating betweenperfectly matched parallel homologous complexes and 1 bp mismatchedparallel homologous complexes.

[0205] Remarkably, perfectly matched antiparallel complementaryssDNA:ssDNA complex formation was enhanced by 48% when Probe No. 3conjugated 25% with thymine was reacted with the sense strand of SEQ IDNO:5 in the presence of YOYO-1 (FIG. 5A). The simultaneous interactionof an adenine in Probe No. 3 with the conjugated complementary thymineand the complementary T in the ssDNA target augmented formation of theperfectly matched antiparallel complementary complex. Remarkably,formation of the 1 bp T-G mismatched antiparallel complementary complexwas very inefficient when T-conjugated Probe No. 3 was used, resultingin an 88% decrease in fluorescent emission intensity compared to thatgenerated by the perfectly matched antiparallel complementary complexcontaining conjugated T (FIG. 5A). It is also remarkable thatdiscrimination between perfectly matched and 1 bp mismatchedantiparallel complementary ssDNA:ssDNA complexes was greatly enhanced byuse of conjugated ssDNA probes in the presence of YOYO-1.

[0206] Twenty-five percent conjugation of Probe No. 3 with cytosine orguanosine also increased the efficiency of both antiparallelcomplementary and parallel homologous ssDNA:ssDNA complex formation inthe presence of YOYO-1, as well as improved the specificity indifferentiation between perfectly matched complexes and 1 bp mismatchedcomplexes (data not shown).

[0207] Formation of ssDNA:ssDNA complexes comprising conjugated basesproves that the bases in a sequence can recognize and interactspecifically and simultaneously with both complementary and homologousbases provided the conjugated base is a Watson-Crick complement to abase on the strand which binds specifically to another strand. Therecognition and binding configurations between bases in a ssDNA probe,conjugated bases and bases in a ssDNA target may be similar to the baseconfigurations formed in antiparallel and parallel dsDNA:ssDNA complexesdescribed herein.

Example 6

[0208] Example 6 demonstrates quadruplex DNA formation between dsDNAtargets containing mixed base sequences and homologous dsDNA probeslabeled with fluorescein. Quadruplex DNA formation is enhanced by thepresence of monovalent cations added to the reaction.

[0209] Complementary sense and antisense 15-mer ssDNA sequences weresynthesized, purified by HPLC and annealed as above to generate 15-merdsDNA probes. DsDNA probes were diluted in ddH₂O at a concentration of 1pmole/μl.

[0210] Probe No. 11 was a 15-mer dsDNA probe with an attachedfluorescein moiety at each 5′ position, and was designed to becompletely homologous to a central 15 bp segment of the 50-mer wild-typetarget DNA (SEQ ID NO:5).

[0211] The sequence for the sense strand of Probe No. 11 (SEQ ID NO:22)was: 5′-Flu-CTG TCA TCT CTG GTG-3′.

[0212] The sequence for the antisense strand of Probe No. 11 (SEQ IDNO:22) was: 5′-Flu-CAC CAG AGA TGA CAG-3′.

[0213] Each hybridization reaction mixture (40 μl) contained thefollowing: 0.4 pmoles of target dsDNA, 4 pmoles of 5′-fluoresceinlabeled dsDNA Probe No. 11, 10 mM Tris-HCl, pH 7.5 and 100 mM KCl. Thereaction mixtures were incubated at room temperature (21° C.) for 1hour, without prior denaturation. Samples were placed into a quartzcuvette, irradiated with an argon ion laser beam having a wavelength of488 nm and monitored for fluorescent emission. The maximum fluorescentintensities occurred at a wavelength of 525 nm, the emission wavelengthfor fluorescein. FIG. 6 shows the intensity of fluorescence plotted as afunction of wavelength for each sample analyzed.

[0214] In the absence of KCl, no binding between the dsDNA targets andProbe No. 11 was detected, resulting in similar fluorescent intensitiesobserved when wild-type dsDNA target (SEQ ID NO:5) or mutant dsDNAtarget (SEQ ID NO:7) were mixed with dsDNA Probe No. 11 or when dsDNAProbe No. 11 was present alone (data not shown).

[0215] After a 1 hour incubation at 21° C. in the presence of 100 mMKCl, dsDNA:dsDNA-F quadruplexes consisting of perfectly homologoussequences on dsDNA target (SEQ ID NO:5) and dsDNA Probe No. 11 formedreadily, resulting in a 62% decrease in the intensity of fluorescentemission compared to that emitted by dsDNA Probe No. 11 alone (labeleddsDNA-F) (FIG. 6). In contrast, incompletely homologous dsDNA:dsDNA-Fquadruplexes (SEQ ID NO:7+Probe No. 11), containing a 1 base pairmismatch were less stable in these reaction conditions, yielding only an18% decrease in fluorescent intensity compared to that exhibited bydsDNA Probe No. 11 alone.

[0216] The presence of monovalent cations, such as K⁺, at specificconcentrations was sufficient to allow quadruplex formation betweendsDNA targets and dsDNA probes labeled with fluorescein in the absenceof prior denaturation. Quadruplex formation occurred on the basis ofhomologous base pair affinities, with a measurable and significantlygreater amount of quadruplex formation between fully homologous duplexstrands. Moreover, the reaction occurred at room temperature within just1 hour of incubation at a ratio of probe to target of 10 to 1, usingnatural dsDNA. The dsDNA targets and dsDNA probe used in this examplewere homologous, contained 53% GC content, and did not containhomopurine or homopyrimidine stretches on any DNA strand. The assay ofthe invention was able to identify perfectly homologous dsDNA sequencesand those containing a pair of mismatched bases, using a dsDNA probe.

Example 7

[0217] The quadruplex DNA assays performed in Example 6 were facilitatedby the addition of monovalent cations in the reaction mixtures. Thespecificity of the assay was further examined utilizing divalent cationsto facilitate quadruplex DNA formation with dsDNA targets and dsDNA-Fprobes possessing 53% GC content.

[0218] Each hybridization reaction mixture (40 μl) contained thefollowing: 0.4 pmoles of target dsDNA, 4 pmoles of 5′-fluoresceinlabeled dsDNA Probe No. 11, 10 mM Tris-HCl, pH 7.5, 20 mM MnCl₂ and 20mM MgCl₂. The reaction mixtures were incubated at room temperature (21°C.) for 1 hour, without prior denaturation. Samples were placed into aquartz cuvette, irradiated with an argon ion laser beam having awavelength of 488 nm and monitored for fluorescent emission. FIG. 7shows the intensity of fluorescence plotted as a function of wavelengthfor each sample analyzed.

[0219] When dsDNA-F Probe No. 11 (with a 53% GC content) was incubatedwith the 50-mer wild-type dsDNA target (SEQ ID NO:5) or the mutant dsDNAtarget (SEQ ID NO:7) in the presence of 20 mM MnCl₂ and 20 mM MgCl₂,quadruplexes were formed at room temperature under non-denaturingconditions. While perfectly homologous DNA quadruplexes yielded themaximum decrease in fluorescent intensity, a 34% decrease, the lessfavourable dsDNA:dsDNA-F quadruplexes containing a 1 bp mismatch (SEQ IDNO:7+Probe No. 11) produced a fluorescent intensity that was about thesame as that observed with dsDNA Probe No. 11 alone (FIG. 7).

[0220] The presence of divalent cations such as Mn⁺² and Mg⁺²facilitated quadruplex formation under non-denaturing conditions toallow accurate discrimination between fully homologous dsDNA target anddsDNA probe quadruplexes, and quadruplex sequences containing a pair ofbases which are mismatched.

Example 8

[0221] The quadruplex DNA assays performed in Examples 6 and 7 werefacilitated by the addition of either monovalent cations or divalentcations in the reaction mixtures. The next Example demonstrates thespecificity of the homologous quadruplex DNA assay when the DNAintercalator, YOYO-1, is employed.

[0222] Complementary sense and antisense 15-mer ssDNA sequences weresynthesized, purified by HPLC and annealed as above to generate 15-merdsDNA probes. DsDNA probes were diluted in ddH₂O at a concentration of 1pmole/μl.

[0223] Probe No. 12 was a 15-mer dsDNA probe identical in sequence toProbe No. 11, but without the attached 5′ fluorescein moieties.

[0224] The sequence for the sense strand of Probe No. 12 (SEQ ID NO:23)was: 5′-CTG TCA TCT CTG GTG-3′.

[0225] The sequence for the antisense strand of Probe No. 12 (SEQ IDNO:23) was: 5′-CAC CAG AGA TGA CAG-3′.

[0226] Each hybridization reaction mixture (40 μl) contained thefollowing: 0.4 pmoles of dsDNA target, 4 pmoles of dsDNA Probe No. 12,0.5×TBE and 100 nM of YOYO-1. The reaction mixtures were incubated at21° C. for 5 minutes, placed into a quartz cuvette, irradiated with anargon ion laser beam having a wavelength of 488 nm and monitored forfluorescent emission. The intensity of fluorescent emission was plottedas a function of wavelength for each sample analyzed.

[0227] The fluorescent intensities observed when no target or probe waspresent (YOYO-1 only) are shown in FIG. 8. FIG. 8 also shows thefluorescent intensities observed when the reaction mixtures combineddsDNA Probe No. 12 with wild-type 50-mer dsDNA target (SEQ ID NO:5)which contained homologous sequences, or with four other dsDNA targetswhich, but for one mismatched pair of bases, contained sequences whichwere homologous to the base sequences in the dsDNA Probe No. 12.Homologous wild-type dsDNA target (SEQ ID NO:5) when present in thereaction mixture with the dsDNA Probe No. 12 produced the greatestfluorescent intensity. Mismatched dsDNA targets when incubated withdsDNA Probe No. 12 in the reaction mixture yielded lesser fluorescentintensity values ranging from 20% less for dsDNA target (SEQ ID NO:10)to 80% less for dsDNA target (SEQ ID NO:11), compared to that achievedby perfectly matched quadruplexes (FIG. 8).

[0228] It was observed that homologous quadruplexes, stabilized byYOYO-1 intercalation, formed more readily between a dsDNA target and adsDNA probe when that probe contained perfectly homologous sequences,than when there was a single pair of bases which were not homologous,that is to say identical, to a pair of bases in the dsDNA target. Thequadruplex complexes described in the foregoing three examples arereferred to by us as mirror homologous.

Example 9

[0229] In this example, 50-mer dsDNA targets were exposed to a 53% GC15-mer dsDNA probe (Probe No. 13), wherein Watson-Crick complementarityexists between bases of the strands of the probe and proximal bases ofthe strands of the target when the major groove of one duplex is placedin the minor groove of the other duplex. The sequences of bases in theduplex probe are not homologous but are inverted in relation to those inthe duplex target. The duplexes, when nested major groove into minorgroove, are parallel to one another, and referred to by us as nestedcomplementary.

[0230] Complementary sense and antisense 15-mer ssDNA sequences weresynthesized, purified by HPLC and annealed as above to generate 15-merdsDNA probes. DsDNA probes were diluted in ddH₂O at a concentration of 1pmole/μl.

[0231] The sequence for the sense strand of Probe No. 13 (SEQ ID NO:24)was: 5′-GAC AGT AGA GAC CAC-3′.

[0232] The sequence for the antisense strand of Probe No. 13 (SEQ IDNO:24) was: 5′-GTG GTC TCT ACT GTC-3′.

[0233] Each hybridization reaction mixture (40 μl) contained thefollowing: 0.4 pmoles of target dsDNA, 4 pmoles of dsDNA Probe No. 13,0.5×TBE and 100 nM of YOYO-1. The reaction mixtures were incubated atroom temperature (21° C.) for 5 minutes, placed in a quartz cuvette,irradiated with an argon ion laser beam having a wavelength of 488 nmand monitored for fluorescent emission. The intensity of fluorescentemission was plotted as a function of wavelength for each sampleanalyzed.

[0234]FIG. 9 illustrates that in the absence of prior denaturation, thehighest fluorescent intensities were achieved when the wild-type 50-merdsDNA target (SEQ ID NO:5) was reacted with the 15-mer dsDNA Probe No.13, which was a perfect match on a nested complementary basis to thedsDNA target (SEQ ID NO:5). The fluorescent intensity is indicative ofDNA binding taking place, in this case quadruplex formation between thedsDNA target and the nested complementary dsDNA probe.

[0235] Mutant dsDNA targets which were mismatched with the duplex probeby a single pair of bases when matching was assessed on the invertedhomology basis of nested complementarity, formed measurably fewerquadruplex complexes with the dsDNA probe, than did the fullycomplementary wild-type dsDNA target. The various mismatches, which wereassayed on a mirror homologous basis in Example 8 were assayed on anested complementary basis in this example.

[0236] As shown in FIG. 9, the fluorescent intensities produced by thequadruplexes formed with the 1 bp mismatched dsDNA targets plus dsDNAProbe No. 13, ranged from 8% to 16% less than that achieved by perfectlymatched quadruplexes (SEQ ID NO:5+Probe No. 13).

[0237] Greater discrimination in fluorescence was observed betweenperfectly homologous and partially homologous quadruplexes in Example 8.This suggests that fully complementary or 1 base pair mismatched nestedcomplementary dsDNA probes bind less discriminately to dsDNA targetsthan do mirror homologous dsDNA probes, which bind with greaterspecificity.

[0238] This example shows that Watson-Crick quadruplex binding betweennested complementary DNA duplexes readily occurs in the presence ofYOYO-1.

Example 10

[0239] Example 10 demonstrates that the assay of the invention candiscriminate between perfectly matched, Watson-Crick complementarydsDNA:ssPNA complexes and dsDNA:ssPNA complexes containing 1 bp, 2 bpand 3 bp mismatches when a cationic decondensing agent, such as the DNAintercalator, YOYO-1 is present.

[0240] Each hybridization reaction mixture (40 μl) contained thefollowing: 2 pmoles of target dsDNA, 2 pmoles of ssPNA probe, 0.5×TBEand 500 nM of YOYO-1. The reaction mixtures were incubated at roomtemperature (21° C.) for 5 minutes, placed into a quartz cuvette,irradiated with an argon ion laser beam having a wavelength of 488 nmand monitored for fluorescent emission. The intensity of fluorescencewas plotted as a function of wavelength for each sample analyzed.

[0241] The fluorescent intensities observed when no DNA or PNA waspresent (YOYO-1 only), or when wild-type SEQ ID NO:1, mutant SEQ ID NO:2or mutant SEQ ID NO:3 were reacted with antiparallel PNA Probe No. 1 orparallel PNA Probe No. 2 are shown in FIGS. 10A and 10B, respectively.DsDNA:ssPNA complexes consisting of perfectly complementary sequences(SEQ ID NO:1+Probe No. 1) allowed maximum interaction between YOYO-1 andthe complexes, yielding the highest fluorescent intensities (FIG. 10A).The fluorescent intensities for a one base pair mismatched dsDNA:ssPNAcomplex (SEQ ID NO:2+Probe No. 1) and a two base pair mismatcheddsDNA:ssPNA complex (SEQ ID NO:3+Probe No. 1) was 97% and 99% lower,respectively, than the perfectly matched dsDNA:ssPNA complex (FIG. 10A).Similarly, when parallel PNA Probe No. 2 was bound to the target dsDNAsequences, the one and two base pair mismatched dsDNA:ssPNA complexesexhibited fluorescent intensities that were 92% and 97% lower,respectively, than the perfectly complementary dsDNA:ssPNA complexes(SEQ ID NO:1+Probe No. 2) (FIG. 10B). Three base pair mismatcheddsDNA:ssPNA complexes consisting of SEQ ID NO:4 and Probe No. 1, or SEQID NO:4 and Probe No. 2 produced fluorescent intensities that were 99%and 97% lower, respectively, than the perfectly matched dsDNA:ssPNAcomplexes (data not shown). Control samples comprising 50-mer dsDNAtargets plus 500 nM YOYO-1 exhibited levels of fluorescence which wereat or below the level of fluorescence observed with 3 bp mismatchedcomplexes (data not shown). The level of fluorescence emitted by eitherssPNA probe plus 500 nM YOYO-1 together was identical to that emitted byYOYO-1 alone (data not shown). As the degree of mismatch between theprobe and the target increased, the level of interaction of YOYO-1 withthe mismatched complexes diminished. Hence the intensity of fluorescentemission decreased. This relationship held whether or not anantiparallel or parallel PNA probe was used. The characteristic level offluorescence emitted by each complex was monitored over time and wasstable between 5 minutes and 24 hours.

[0242] Interestingly, when 15-mer target dsDNA sequences were reactedwith 15-mer PNA probe sequences, larger differences in fluorescentemission were observed between perfectly matched complexes and 1 or 2 bpmismatched complexes when parallel PNA probes were used, than whenantiparallel PNA probes were used (data not shown).

[0243] Therefore, the fluorescent intensity assay measuring dsDNA:ssPNAcomplex formation is able to distinguish between wild-type sequences andthose containing 1 bp, 2 bp or 3 bp mutations, without priordenaturation of the duplex DNA target.

Example 11

[0244] The specificity of the assay measuring triplex formation promotedby YOYO-1 was further investigated by reacting wild-type and mutantdsDNA targets of mixed base sequence with antiparallel and parallelssDNA probes in the absence of prior denaturation of dsDNA targets.

[0245] Each hybridization reaction mixture (40 μl) contained thefollowing: 2 pmoles of target dsDNA, 2 pmoles of ssDNA probe, 0.5×TBEand 500 nM of YOYO-1. The reaction mixtures were incubated at roomtemperature (21° C.) for 5 minutes, placed into a quartz cuvette,irradiated with an argon ion laser beam having a wavelength of 488 nmand monitored for fluorescent emission. The intensity of fluorescencewas plotted as a function of wavelength for each sample analyzed.

[0246] When the antiparallel ssDNA Probe No. 3 (with a 53% GC content)was reacted with the 50-mer wild-type dsDNA target (SEQ ID NO:5) andmutant dsDNA targets (SEQ ID NO:6 and SEQ ID NO:8), dsDNA:ssDNAcomplexes were formed at room temperature under non-denaturingconditions (FIG. 11A). While perfectly matched DNA complexes emitted thehighest fluorescent intensities, incompletely complementary complexeswith a 1 bp mismatch (SEQ ID NO:6+Probe No. 3) and a consecutive 2 bpmismatch (SEQ ID NO:8+Probe No. 3) produced fluorescent intensities thatwere 63% and 95% lower, respectively, than that observed with theperfectly matched sequences (FIG. 11A). The level of fluorescencediminished as the degree of mismatch between the probe and targetincreased. The characteristic fluorescent intensity exhibited by eachcomplex was monitored over time and was stable between 5 minutes and 24hours. Control samples comprising 50-mer dsDNA targets plus 500 nMYOYO-1 exhibited levels of fluorescence which were below the level offluorescence observed with 2 bp mismatched DNA complexes (data notshown). The level of fluorescence generated by the ssDNA probe plus 500nM YOYO-1 was identical to that achieved by YOYO-1 alone (data notshown). Very similar results were obtained when 15-mer antiparallelssDNA probes were reacted with wild-type or mutant 50-mer dsDNA targetshaving 33% GC and 73% GC contents under the same reaction conditions,demonstrating the reliability of the dsDNA:ssDNA complex formation assayutilizing antiparallel ssDNA probes, independent of the percent GCcontent of the ssDNA probes and dsDNA targets (data not shown).

[0247] Similarly, in the presence of YOYO-1, dsDNA:ssDNA complexes wereformed when the parallel ssDNA Probe No. 4 was reacted with the 50-merwild-type dsDNA target (SEQ ID NO:5) and mutant dsDNA targets (SEQ IDNO:6 and SEQ ID NO:8). The fluorescent intensities for a 1 bp mismatchedDNA complex (SEQ ID NO:6+Probe No. 4) and a consecutive 2 bp mismatchedDNA complex (SEQ ID NO:8+Probe No. 4) were 48% and 65% lower,respectively, than that obtained by the perfectly matched sequences(FIG. 11B). As the degree of mismatch between the probe and targetincreased, the level of fluorescent emission decreased. Slightly lessdiscrimination between perfectly matched and mismatched DNA complexeswas observed when a parallel ssDNA probe was used than when anantiparallel ssDNA probe was used to generate the dsDNA:ssDNA complexes.

[0248] YOYO-1 facilitated DNA complex formation between an antiparallelssDNA probe and dsDNA targets, and between a parallel ssDNA probe anddsDNA targets, to allow differentiation between perfectly matchedcomplexes and those containing 1 bp or 2 bp mismatches, without therequirement for prior denaturation of dsDNA targets.

Example 12

[0249] The complexes formed in Examples 10 and 11 were stabilized by theDNA intercalator, YOYO-1 present in the reaction mixtures. Thespecificity of the assay was further examined utilizing divalent cationsto promote and stabilize complex formation with dsDNA targets andssDNA-F probes.

[0250] Each hybridization reaction mixture (40 μl) contained thefollowing: 0.4 pmoles of target dsDNA, 4 pmoles of 5′-fluoresceinlabeled ssDNA probe, 10 mM Tris-HCl, pH 7.5, and 1 mM to 20 mM each ofMgCl₂ and MnCl₂. The reaction mixtures were incubated at roomtemperature (21° C.) for various lengths of time ranging from 1 minuteto 2 hours, without prior denaturation of dsDNA targets. Followingincubation, samples were placed into a quartz cuvette, irradiated withan argon ion laser beam having a wavelength of 488 nm and monitored forfluorescent emission. The maximum fluorescent intensities occurred at awavelength of 525 nm, the emission wavelength for fluorescein. Theintensity of fluorescent emission was plotted as a function ofwavelength for each sample analyzed.

[0251] When the antiparallel ssDNA-F Probe No. 5 was incubated for 1hour with the 50-mer wild-type dsDNA target (SEQ ID NO:5) in thepresence of 15 mM MgCl₂ and 15 mM MnCl₂, perfectly complementarydsDNA:ssDNA-F complexes were formed very efficiently, generating a 74%decrease in fluorescence compared to that achieved by Probe No. 5 alone(FIG. 12A). By contrast, dsDNA:ssDNA-F complexes that contained a 1 bpT-G mismatch (SEQ ID NO:7+Probe No. 5) were much less stable in thepresence of 15 mM MgCl₂ and 15 mM MnCl₂, yielding a 15% decrease influorescence compared to that emitted by Probe No. 5 alone after a 1hour incubation (FIG. 12A). When Probe No. 5 (containing a 53% GCcontent) was reacted with the dsDNA target SEQ ID NO:9 (containing a 33%GC content), a 3% increase in fluorescence was observed compared to thatobtained by Probe No. 5 alone (FIG. 12A), indicative of no DNA complexformation. This result was expected considering this probe and targetcombination would result in a 5 bp mismatch.

[0252] In the presence of 10 mM MgCl₂ and 10 mM MnCl₂, the dsDNA:ssDNA-Fcomplexes possessing a 53% GC content and containing perfectlycomplementary sequences (SEQ ID NO:5+Probe No. 5) or a 1 bp T-G mismatch(SEQ ID NO:7+Probe No. 5) generated fluorescent intensities that were68% and 20% lower, respectively, after an 1 hour incubation, and 76% and16% lower, respectively, after a 30 minute incubation, than that emittedby Probe No. 5 alone (data not shown). The addition of 5 mM MgCl₂ and 5mM MnCl₂ (or lower concentrations) was insufficient to allow complexformation between the antiparallel ssDNA-F Probe No. 5 and all dsDNAtargets tested following a 1 hour incubation (data not shown).

[0253] DsDNA:ssDNA complexes were also formed when the parallel ssDNAProbe No. 6 was reacted with the 50-mer wild-type dsDNA target (SEQ IDNO:5) and mutant dsDNA target (SEQ ID NO:7) In this case DNA complexformation was promoted with much lower concentrations of MgCl₂ and MnCl₂(i.e. 1-5 mM each) requiring shorter incubation periods. Incubation inthe presence of 1 mM MgCl₂ and 1 mM MnCl₂, or 2 mM MgCl₂ and 2 mM MnCl₂for 15 minutes was sufficient to generate DNA complexes (data notshown). The fluorescent intensities for a perfectly matched DNA complex(SEQ ID NO:5+Probe No. 6) and a 1 bp mismatched DNA complex (SEQ IDNO:7+Probe No. 6) were 29% and 6% lower, respectively, than thatobtained by parallel ssDNA Probe No. 6 alone in the presence of 3 mMMgCl₂ and 3 mM MnCl₂ after a 45 minute incubation (FIG. 12B)

[0254] Although DNA complexes formed readily at 10 mM MgCl₂ and 10 mMMnCl₂ after a 1 hour incubation, no discrimination between perfectlymatched and mismatched complexes was observed when a parallel ssDNAprobe was used. Concentrations above 15 mM MgCl₂ and 15 mM MnCl₂ wereinhibitory for DNA complex formation with a parallel ssDNA probe (datanot shown).

[0255] The addition of salt bridging, condensing agents such as divalentcations promoted DNA complex formation between non-denatured dsDNAtargets and fluorescently-labeled antiparallel or parallel ssDNA probes,to allow accurate and reliable discrimination between perfectlycomplementary sequences and those containing 1 bp mutations. Thereactions occurred at room temperature within 15-60 minutes ofincubation at a ratio of probe to target of 10 to 1. The dsDNA targetsand ssDNA probes did not contain homopurine or homopyrimidine stretchesof DNA. Despite the presence of 5 pyrimidine bases interspersed withinthe 15 nucleotide ssDNA probes, DNA complexes formed readily in asequence specific manner.

Example 13

[0256] The utility of probes of varying directionality was alsoevaluated when monovalent cations were employed to promote and stabilizecomplex formation with dsDNA targets.

[0257] Each hybridization reaction mixture (40 μl) contained thefollowing: 0.4 pmoles of target dsDNA, 4 pmoles of 5′-fluoresceinlabeled ssDNA probe, 10 mM Tris-HCl, pH 7.5, and 10 mM to 150 mM NaCl.The reaction mixtures were incubated at room temperature (21° C.) forvarious lengths of time ranging from 1 minute to 2 hours, without priordenaturation of dsDNA targets. Following incubation, samples were placedinto a quartz cuvette, irradiated with an argon ion laser beam having awavelength of 488 nm and monitored for fluorescent emission. The maximumfluorescent intensities occurred at a wavelength of 525 nm, the emissionwavelength for fluorescein. The intensity of fluorescent emission wasplotted as a function of wavelength for each sample analyzed.

[0258] In the absence of NaCl or presence of 10 mM or 25 mM NaCl, nobinding between the dsDNA targets (SEQ ID NO:1 or SEQ ID NO:2) and theantiparallel ssDNA-F Probe No. 7 was detected, after all incubationperiods (data not shown).

[0259] After a 1 hour incubation in the presence of 50 mM NaCl,dsDNA:ssDNA-F complexes consisting of perfectly complementary sequences(SEQ ID NO:1+Probe No. 7) formed readily, resulting in a 49% decrease influorescent emission intensity compared to that emitted by the controlProbe No. 7, which was similarly incubated in the reaction mixture (FIG.13A). By contrast, incompletely complementary dsDNA:ssDNA-F complexescontaining a 1 bp G-T mismatch (SEQ ID NO:2+Probe No. 7) yielded a 11%decrease in fluorescent emission intensity compared to that exhibited bythe Probe No. 7 control sample.

[0260] The presence of 75 mM, 100 mM and 125 mM NaCl in the reactionmixture also resulted in fluorescent emission quenching consistent withsignificant amounts of complex formation between the perfectly matchedSEQ ID NO:1 target and antiparallel Probe No. 7, and significantly lessquenching when the 1 bp G-T mismatched SEQ ID NO:2 target and Probe No.7 were present, producing similar fluorescent intensities to thatobserved in the presence of 50 mM NaCl (data not shown).

[0261] DsDNA:ssDNA complexes were also formed when the parallel ssDNAProbe No. 6 was reacted with the 50-mer wild-type dsDNA target (SEQ IDNO:5) and mutant dsDNA target (SEQ ID NO:7) in the presence of 50 mM, 75mM, 100 mM or 150 mM NaCl. Optimum results were obtained in the presenceof 100 mM NaCl. After a 75 minute incubation at room temperature in areaction mixture containing 100 mM NaCl, the fluorescent emissionintensities for a perfectly matched DNA complex (SEQ ID NO:5+Probe No.6) and a 1 bp mismatched DNA complex (SEQ ID NO:7+Probe No. 6) were 53%and 9% lower, respectively, than that obtained by the control parallelssDNA Probe No. 6 reacted under the same conditions (FIG. 13B). 50 mMNaCl promoted maximum discrimination between perfectly matched andmismatched complexes in an incubation period of 45 minutes (data notshown). In general, complexes containing either antiparallel or parallelssDNA probes seemed to form with similar efficiencies at similar NaClconcentrations and incubation periods.

[0262] Use of monovalent cations, which are known DNA condensing agents,facilitated DNA complex formation between non-denatured dsDNA targetsand fluorescently-labeled antiparallel or parallel ssDNA probes, toallow reliable differentiation between complexes containing perfectlycomplementary sequences and those containing 1 bp mismatches.

Example 14

[0263] DsDNA:ssDNA complexes facilitated by YOYO-1 readily form at roomtemperature within 5 minutes of incubation and generate fluorescentemissions at the same level of intensity for hours. Complexes containingbase pair mismatches similarly emit fluorescent signals which persist,indicating the same level of complex formation over time. To examine therate of formation, stability and rate of disassociation of dsDNA:ssDNAcomplexes formed in the presence of condensing agents such as cations,time course experiments were performed.

[0264] Each hybridization reaction mixture (40 μl) contained thefollowing: 0.4 pmoles of non-denatured target dsDNA, 4 pmoles of5′-fluorescein labeled ssDNA probe, 10 mM Tris-HCl, pH 7.5, 10 mM MgCl₂and 10 mM MnCl₂. The reaction mixtures were incubated at roomtemperature (21° C.) for various periods ranging from 1 minute to 2hours. Following incubation, samples were placed into a quartz cuvette,irradiated once with an argon ion laser beam having a wavelength of 488nm and monitored for fluorescent emission. Further fluorescentmeasurements were taken of the same samples after subsequent multiplelaser irradiation, at the indicated times (FIG. 14). The intensity offluorescence was plotted as a function of time for each sample analyzed.

[0265] The fluorescence emitted by control samples comprising 4 pmolesof Probe No. 5 plus 10 mM MgCl₂ and 10 mM MnCl₂, in the absence oftarget dsDNA, dramatically decreased 3-fold within just 5 minutes ofincubation (data not shown), and then steadily declined at a much slowerrate within the next few hours (FIG. 14A). This effect we refer to as“Cationic Quench”. This inhibition of fluorescence, associated withincreased incubation periods of ssDNA-F probes with specific cations,occurred routinely in the presence of divalent cations, but not in thepresence of monovalent cations (data not shown). This observation makesevident the importance of incubating the control sample in an experimentunder exactly the same conditions that the test samples of an experimentare reacted. Multiple lasing of each ssDNA-F control sample aftervarying periods of incubation inhibited further quenching of thefluorophore, resulting in a steady level of fluorescence thereafter(FIG. 14A). This result was entirely unanticipated.

[0266] When the antiparallel ssDNA-F Probe No. 5 was incubated with the50-mer wild-type dsDNA target (SEQ ID NO:5) in the presence of 10 mMMgCl₂ and 10 mM MnCl₂, dsDNA:ssDNA-F complex formation was evident after15 minutes of incubation resulting in a decrease in fluorescence, whichwas 6% greater than the progressive cationic quench of the control ProbeNo. 5 (compare FIGS. 14A and 14B). Complex formation was greatlyindicated after 30 and 60 minutes of incubation of SEQ ID NO:5 withProbe No. 5 in the presence of 10 mM MgCl₂ and 10 mM MnCl₂, generating a76% and 61% decrease in fluorescence, respectively, compared to thatachieved by the cationically quenched Probe No. 5 alone (FIG. 14B).After 90 and 120 minutes of incubation in the presence of 10 mM MgCl₂and 10 mM MnCl₂, no complex formation was being signaled (FIG. 14B). Thelevel of fluorescent emission seen at 90 and 120 minutes was whollyattributable to the cationic quench effect (compare FIGS. 14A and 14B).

[0267] By contrast, dsDNA:ssDNA-F complexes that contained a 1 bp T-Gmismatch (SEQ ID NO:7+Probe No. 5) formed at a slower rate and were muchless stable once formed in the presence of 10 mM MgCl₂ and 10 mM MnCl₂.The 1 bp T-G mismatched complex was first observed after 30 minutes ofincubation, and appeared to have been eliminated after 60 minutes ofincubation (FIG. 14C). Once again, the probe was antiparallel to thecomplementary strand in the duplex (FIG. 14C).

[0268] Multiple laser irradiation of perfectly complementary dsDNA:ssDNAcomplexes (SEQ ID NO:5+Probe No. 5) formed after 30 minutes or 60minutes of incubation in the presence of 10 mM MgCl₂ and 10 mM MnCl₂resulted in fluorescent emissions consistent with the destruction ofthese complexes at a rate characteristic for DNA complexes containing anantiparallel ssDNA probe (FIG. 14B). When a subsequent measurement wasmade at 45 minutes after lasing of the perfectly complementary complexat 30 minutes, the emission intensity level was 1869, testimony to therapidity with which the complex was destroyed (data not shown). Thelevel of fluorescent emission, after multiple lasing, returned to thecationically quenched values observed by the uncomplexed Probe No. 5alone control (compare FIGS. 14A and 14B). The only exception was theperfectly matched complexes formed after 15 minutes of incubation andrepeatedly irradiated thereafter (FIG. 14B). In this case thefluorescent emission was not consistent with the destruction of thecomplexes (FIG. 14B), even though further cationic quench of Probe No.5, when multiply irradiated after a 15 minute incubation, was totallyinhibited (FIG. 14A). DsDNA:ssDNA complexes containing a 1 bp T-Gmismatch (SEQ ID NO:7+Probe No. 5) were similarly apparently destroyedby multiple lasing (FIG. 14C).

[0269] An experiment was performed to determine the basis for the effectof multiple lasing on the complexes. It was found that when freshcations were added to the reaction mixture which had been lased twice,the inhibition of cationic quench in fluorescence emitted by the ssDNA-Fprobe could not be reversed and further cationic quench did not occurupon further incubation, strongly suggesting that the ssDNA-F probe wasinactivated by multiple irradiation, by a yet unknown mechanism (datanot shown). Similarly, when fresh ssDNA-F probes were added to thereaction mixture which had been lased twice, after normalizing for theincreased fluorescent emission of the fresh probe, no subsequentprogressive cationic quenching was observed upon further incubation ofthe reaction mixture, strongly suggesting that the lased cations weresomehow disabled (data not shown).

Example 15

[0270] Clear Lysate

[0271] A concern raised by the use of plasmid viral DNA or viral DNA asa gene therapy vector or as a DNA vaccine is the purity of the DNA.Current large-scale purification methods such as ultracentrifugation inCsCl gradients, glass fiber filters or chromatography can be inefficientin removing contaminants such as host genomic DNA and RNA or proteins.Particularly, host genomic DNA whose chemical structure is very close tothat of plasmid/BAC/PAC DNA is extremely difficult to remove usingclassical chromatography. Typical concentrations of up to 0.5 to 1% hostgenomic DNA are found in preparations obtained by classical methods.Therefore, in order to develop DNA as a safe vector for human genetherapy, there is a need for purification technologies that will lowerthe content of host genomic DNA down to much lower levels.

[0272] PCR products are generally used for microarray spotting contentrather than plasmid, primarily due to the fact that autofluorescentcontaminating materials in plasmid preparations make spotted plasmidunusable. There is a need for purification technologies that will lowerthe content of protein/lipopolysaccharide in plasmid DNA down to muchlower levels.

[0273] Prospective Examples 15 and 16 demonstrate how the presentinvention will address the foregoing problems.

[0274] Plasmid/BAC/PAC DNA is purified from a clear lysate of bacterialculture, on the so-called “miniprep” scale: 1.0 ml of an overnightculture of DH5α strains containing plasmid/BAC/PAC are centrifuged, andthe pellet is resuspended in 160 μl of 30 mM glucose, 15 mM Tris-HCl, pH8, 30 mM EDTA, 100 μg/ml RNAse A. Cells are vortexed for 2 minutes. 160μl of fresh 0.2 M NaOH, 1% SDS are added, the plates are inverted tomix, 160 μl of 3.6 M potassium acetate, 6 M Acetic acid are then addedand the tubes are inverted to mix 5 or 6 times. After centrifugation,the supernatant is recovered and loaded onto an oligonucleotide well asdescribed above and shown in FIGS. 16-17, with biotin-oligo-dT. Bindingbuffer conditions are identical to those required for triplex formationin solution described above. Five washes are performed withhybridization buffer described above, and gentle elution is performedwith 10 mM Tris-HCl, pH 7.5, to dissociate the triplex. Theplasmid/BAC/PAC obtained, analyzed by agarose gel electrophoresis andethidium bromide staining, will take the form of a single band of“supercoiled” circular DNA. No trace of high molecular weight(chromosomal) DNA or of RNA should be detectable in the plasmid/BAC/PACpurified by this method. The ratio of the optical densities at 260 and280 nm should be greater than or equal to 2.

Example 16

[0275] Miniprep-purified Plasmid

[0276] We will purify plasmid/BAC/PAC DNA from a clear lysate ofbacterial culture, on the so-called “miniprep” scale: 1.0 ml of anovernight culture of DH5α strains containing plasmid/BAC/PAC arecentrifuged, and the pellet is resuspended in 160 μl of 30 mM glucose,15 mM Tris-HCl, pH 8, 30 mM EDTA, 100 μg/ml RNAse A. Cells are vortexedfor 2 minutes. 160 μl of fresh 0.2 M NaOH, 1% SDS are added, the platesare inverted to mix, 160 μl of 3.6 M potassium acetate, 6 M Acetic acidare then added and the tubes are inverted to mix 5 or 6 times. Aftercentrifugation, 220 μl of the supernatant is recovered and loaded onto aMillipore FB glass fiber filter plate (pre-loaded with 150 μl of 5.3 MGuanidine-HCl). After mixing, a vacuum is applied for 3 minutes slowlydrawing the liquid through the plate. Next 200 μl of 100% ethanol areadded to each well. A vacuum is applied as above, followed by blottingto remove all the ethanol and drying—no residual ethanol is acceptable.The plasmid is dissolved in 65 μl ddH₂O, incubated at room temperaturefor 5 mins and transferred from the Millipore FB plate into a 96 wellcollection plate by centrifugation at 1000×g for 5 minutes. Using thisprep, (35-125 ng/μl) the collected plasmid is recovered and loaded ontoan oligonucleotide well as described above with biotin-oligodT. Bindingbuffer conditions are identical to those required for triplex formationin solution described above. Five washes are performed withhybridization buffer described above, and gentle elution is performedwith 10 mM Tris-HCl, pH 7.5, to dissociate the triplex. Theplasmid/BAC/PAC obtained, analyzed by agarose gel electrophoresis andethidium bromide staining, will take the form of a single band of“supercoiled” circular DNA. No trace of high molecular weight(chromosomal) DNA or of RNA should be detectable in the plasmid/BAC/PACpurified by this method. The ratio of the optical densities at 260 and280 nm should be greater than or equal to 2.

[0277] Removal of the approximately 1% of chromosomal DNA in the sampleapplied to the well would mean that this purification technology willlower the content of host genomic DNA down to much lower levels andallow DNA such as a BAC as a safe vector for human gene therapy.

Example 17

[0278] Plasmid Purification Based on Insert Sequence

[0279] We will purify from a mixture of two plasmids only one plasmidbased on the sequence specificity of the insert. The two plasmids aredissolved in 80 μl of hybridization buffer (10 mM Tris-HCl, pH 7.5, 15mM MnCl₂ and 15 mM MgCl₂) and loaded onto the streptavidin agarosecolumn as described above with a 50-mer biotin-oligonucleotide specificto only one plasmid of the two. Loading the hybridization solution ontothe column, loading an additional 3 ml of hybridization buffer onto thecolumn, and passing the elution volume through the column three timesperforms binding. The settled matrix is washed with 5×1 ml hybridizationbuffer, and the desired plasmid is eluted with 3×1 ml 10 mM Tris-HCl, pH7.5. The purified plasmid is analyzed by agarose gel electrophoresis andethidium bromide staining, and will take the form of a single band ofplasmid DNA, with no trace of the second plasmid (a second band)detectable. Repeating the protocol with a streptavidin agarose column asdescribed above with a 50-mer biotin-oligonucleotide specific to thesecond plasmid will show a single band of plasmid DNA at the appropriatesize for the second plasmid, with no trace of the first plasmid.

[0280] In the foregoing examples and description, we have elucidatedthat heteropolymeric nucleic acid strands can specifically bind on thebasis of homologous base pairing. Such binding can occur betweenparallel or antiparallel strands.

[0281] We have also elucidated that nucleic acid bases bound in aWatson-Crick complementary duplex are not quiescent as regards the basesof proximal nucleic acid strands and that such bases can be interactedwith on the basis of Watson-Crick complementary base pairing orhomologous base pairings, depending on the binding potential of theproximal sequence of bases determined by either of the possible bindingmotifs. This is true whether the bases in the duplex are interactingwith bases in a third strand to form a specifically bound triplexstructure or whether the bases of the duplex are specificallyinteracting with proximal bases which are themselves coupled into aWatson-Crick complementary duplex. Accordingly the invention comprisesthe discovery that Watson-Crick coupled bases remain reactive asspecific bases to interact and bind to proximal bases on other strandsand do so with great specificity and alacrity. While all of this isremarkable, it is considered especially remarkable that A:T and G:Cpairings are detected as mismatches in binding reactions wherein thehomologous binding motif is dominant and being enforced on all basepairs by a strand-wide imperative. It is likewise remarkable thathomologous quadruplex binding is more specific than is Watson-Crickcomplementary quadruplex binding. Of necessity quadruplex binding occursbetween the major groove side of a duplex-coupled base or base pair andthe minor groove side of a duplex-coupled base or base pair. Heretofore,while the potential of further binding by a base already complexed in aduplex was unknown, it had been postulated that third strand recognitionof bases in a duplex occurred solely in the major groove of the duplex.This we show is not the case. We have also demonstrated that putativebackbone repulsion is no barrier to duplex:duplex interaction.

[0282] Our invention relates to readily achieved binding reactions whichare typically achieved with short incubation periods at room temperatureand which do not depend on molar excess of a reagent to drive areaction. Accordingly the invention is shown to be not only readilyachieved, but obviously biologically relevant.

[0283] Finally we have shown that partial Watson-Crick complementaryconjugation with free bases can contribute to increased duplex bindingand increased specificity.

[0284] The invention constitutes a substantial addition to the knowledgeof base binding and as such will be central to the elucidation of manybiological functions whose mechanisms are currently mysterious, such asgene silencing.

[0285] It is most remarkable to detect specific homologous recognitionand binding by bases previously and stably coupled into Watson-Crickcomplementary duplex.

[0286] We believe that what we have elucidated will require theabandonment of many “canonical” ideas and the reopening of the questionof nucleic acid binding capability.

[0287] While the invention has been described in detail and withreference to specific examples thereof, it will be apparent to oneskilled in the art that various changes and modifications can be madetherein without departing from the spirit and scope thereof.

1 24 1 50 DNA Exon 10 of Human Cystic Fibrosis Gene 1 tggcaccattaaagaaaata tcatctttgg tgtttcctat gatgaatata 50 2 50 DNA Exon 10 of HumanCystic Fibrosis Gene 2 tggcaccatt aaagaaaata tcgtctttgg tgtttcctatgatgaatata 50 3 50 DNA Exon 10 of Human Cystic Fibrosis Gene 3tggcaccatt aaagaaaata tactctttgg tgtttcctat gatgaatata 50 4 50 DNA Exon10 of Human Cystic Fibrosis Gene 4 tggcaccatt aaagaaaata tacgctttggtgtttcctat gatgaatata 50 5 50 DNA Exon 10 of Human Cystic Fibrosis Gene5 gagcaccatg acagacactg tcatctctgg tgtgtcctac gatgactctg 50 6 50 DNAExon 10 of Human Cystic Fibrosis Gene 6 gagcaccatg acagacactg tcgtctctggtgtgtcctac gatgactctg 50 7 50 DNA Exon 10 of Human Cystic Fibrosis Gene7 gagcaccatg acagacactg tcatctttgg tgtgtcctac gatgactctg 50 8 50 DNAExon 10 of Human Cystic Fibrosis Gene 8 gagcaccatg acagacactg tactctctggtgtgtcctac gatgactctg 50 9 47 DNA Exon 10 of Human Cystic Fibrosis Gene9 tggcaccatt aaagaaaata tcattggtgt ttcctatgat gaatata 47 10 50 DNA Exon10 of Human Cystic Fibrosis Gene 10 gagcaccatg acagacactg tcttctctggtgtgtcctac gatgactctg 50 11 50 DNA Exon 10 of Human Cystic Fibrosis Gene11 gagcaccatg acagacactg tcatccctgg tgtgtcctac gatgactctg 50 12 15 DNAExon 10 of Human Cystic Fibrosis Gene misc_feature ()..() 5′ end beginswith H, 3′ end ends with lysine-CONH2 12 caccaaagat gatat 15 13 15 DNAExon 10 of Human Cystic Fibrosis Gene misc_feature ()..() 5′ end beginswith H, 3′ end ends with lysine-CONH2 13 tatagtagaa accac 15 14 15 DNAExon 10 of Human Cystic Fibrosis Gene 14 caccagagat gacag 15 15 15 DNAExon 10 of Human Cystic Fibrosis Gene 15 gacagtagag accac 15 16 15 DNAExon 10 of Human Cystic Fibrosis Gene misc_feature ()..() fluoresceinmoiety attached at the 5′ position 16 caccagagat gacag 15 17 15 DNA Exon10 of Human Cystic Fibrosis Gene misc_feature ()..() fluorescein moietyattached at 5′ end 17 gacagtagag accac 15 18 15 DNA Exon 10 of HumanCystic Fibrosis Gene misc_feature ()..() fluorescein moiety attached at5′ end 18 caccaaagat gatat 15 19 15 DNA Exon 10 of Human Cystic FibrosisGene 19 caccaaagat gatat 15 20 15 DNA Exon 10 of Human Cystic FibrosisGene 20 cacgaaagat gatat 15 21 15 DNA Exon 10 of Human Cystic FibrosisGene 21 caccaaacat gatat 15 22 15 DNA Exon 10 of Human Cystic FibrosisGene misc_feature ()..() fluorescein moiety attached at 5′ end 22ctgtcatctc tggtg 15 23 15 DNA Exon 10 of Human Cystic Fibrosis Gene 23ctgtcatctc tggtg 15 24 15 DNA Exon 10 of Human Cystic Fibrosis Gene 24gacagtagag accac 15

What is claimed is:
 1. A method for capturing and separating a targetfrom a sample, said method comprising: providing the sample comprisingthe target, wherein the target contains a heteropolymeric targetsequence of nucleic acids or nucleic acid analogues; providing a probecomprising a heteropolymeric probe sequence of nucleic acids or nucleicacid analogues; bonding the heteropolymeric target sequence to theheteropolymeric probe sequence to provide a complex; attaching the probeto a support before, during or after the complex is provided; andseparating the sample from the complex to separate the target from thesample, wherein the heteropolymeric probe sequence is bonded to theheteropolymeric target sequence by Watson-Crick complementary baseinteraction or by homologous base interaction, provided that when thecomplex is a duplex and the heteropolymeric probe sequence isantiparallel to the heteropolymeric target sequence, the heteropolymericprobe sequence is bonded to the heteropolymeric target sequence byhomologous base interaction, and provided that when the complex is atriplex, the complex is free of recombination proteins.
 2. The method ofclaim 1, wherein the probe and the target are single-stranded and thecomplex is a duplex wherein the heteropolymeric probe sequence is bondedto the heteropolymeric target sequence by homologous base interaction orby Watson-Crick complementary base interaction and the heteropolymericprobe sequence is parallel or antiparallel to the heteropolymeric targetsequence.
 3. The method of claim 1, wherein one of the probe and thetarget is single-stranded, the other of the probe and the target isdouble-stranded, and the complex is a triplex.
 4. The method of claim 1,wherein the probe and the target are double-stranded and the complex isa quadruplex.
 5. The method of claim 1, wherein at least one of theprobe and the target is double-stranded and the complex is a triplex ora quadruplex.
 6. The method of claim 1, wherein the complex isimmobilized by the support, and the separating of the sample from thecomplex comprises washing the sample, the immobilized complex and thesupport with a liquid.
 7. The method of claim 6, wherein the support isa bead, a plate, a membrane or a film.
 8. The method of claim 7, whereinthe probe contains biotin, the support is at least partially coated withstreptavidin and the complex is attached to the support by linking thebiotin to the streptavidin.
 9. The method of claim 1, wherein the samplefurther comprises at least one member selected from the group consistingof proteins, lipopolysaccharides, nucleases and additional nucleic acidsequences other than the heteropolymeric target sequence.
 10. The methodof claim 1, wherein the probe invades the target, displacing a sequenceof bases bound to the heteropolymeric target sequence, such that basesof the heteropolymeric probe sequence bind with bases of theheteropolymeric target sequence on the basis of Watson-Crick baserecognition or homologous base recognition.
 11. The method of claim 1,wherein the method is conducted without denaturing the probe or thetarget.
 12. The method of claim 1, wherein the sample is contacted withthe target in a liquid medium further comprising at least one bindingpromoter.
 13. The method of claim 12, wherein the at least one bindingpromoter is a condensing agent or a decondensing agent.
 14. The methodof claim 12, wherein the at least one binding promoter is a memberselected from the group consisting of YOYO-1, TOTO-1, YOYO-3, TOTO-3,POPO-1, BOBO-1, POPO-3, BOBO-3, LOLO-1, JOJO-1, cyanine dimers,YO-PRO-1, TO-PRO-1, YO-PRO-3, TO-PRO-3, TO-PRO-5, PO-PRO-1, BO-PRO-1,PO-PRO-3, BO-PRO-3, LO-PRO-1, JO-PRO-1, cyanine monomers, ethidiumbromide, ethidium homodimer-1, ethidium homodimer-2, ethidiumderivatives, acridine, acridine orange, acridine derivatives,ethidium-acridine heterodimer, ethidium monoazide, propidium iodide,SYTO dyes, SYBR Green 1, SYBR dyes, Pico Green, SYTOX dyes and7-aminoactinomycin D.
 15. The method of claim 1, further comprisingseparating the target from the probe after the sample has been separatedfrom the complex.
 16. The method of claim 1, wherein the target isbrought into proximity with the probe by moving the sample over or pastthe support at least once.
 17. The method of claim 1, carried out insolution, on a biochip, in a capillary, in a microwell channel or in acolumn.
 18. A kit for performing the method of claim 1, the kitcomprising the support and the probe, wherein the probe is attached tothe support or adapted to attach to the support during performance ofthe method, and wherein the probe is adapted to form the complex withthe target.
 19. The kit of claim 18, wherein the support is a bead, aplate, a membrane, a film, a microwell, a column or a capillary tube.20. The kit of claim 19, wherein the probe contains biotin and thesupport is at least partially coated with streptavidin to which thebiotin is bound or adapted to be bound.
 21. The kit of claim 20, whereinthe probe is double-stranded.
 22. The kit of claim 18, furthercomprising at least one binding promoter selected from the groupconsisting of YOYO-1, TOTO-1, YOYO-3, TOTO-3, POPO-1, BOBO-1, POPO-3,BOBO-3, LOLO-1, JOJO-1, cyanine dimers, YO-PRO-1, TO-PRO-1, YO-PRO-3,TO-PRO-3, TO-PRO-5, PO-PRO-1, BO-PRO-1, PO-PRO-3, BO-PRO-3, LO-PRO-1,JO-PRO-1, cyanine monomers, ethidium bromide, ethidium homodimer-1,ethidium homodimer-2, ethidium derivatives, acridine, acridine orange,acridine derivatives, ethidium-acridine heterodimer, ethidium monoazide,propidium iodide, SYTO dyes, SYBR Green 1, SYBR dyes, Pico Green, SYTOXdyes and 7-aminoactinomycin D.